Seurat smart seq2

Seurat smart seq2. Overall, Smart-seq2 detected the most genes per cell, while the other methods displayed less amplification noise due to the use of UMIs. Smart-seq2 [7] and 10X Genomics Chromium (10X; 10X Genomics, Pleasanton, CA) are two frequently-used scRNA-seq platforms ( Figure 1 A) [8], [9]. If you use Seurat in your research, please considering Apr 21, 2023 · CELLxGENE Census - Integrating multi-dataset slices of data with Seurat; by Pablo Garcia; Last updated about 1 year ago Hide Comments (–) Share Hide Toolbars Nov 29, 2021 · Single-cell or 100 cells (bulk P100) were sorted for library construction following the Smart-seq2 protocol. The authors performed 457 optimisation experiments to test conditions. Mar 10, 2020 · “harmony”整合不同平台的单细胞数据之旅. Smart-seq2. Dec 2, 2021 · The Smart-Seq2 scRNA-Seq data allowed isoform quantification to be conducted, since full-length transcript sequencing of single cells is performed in this method (Picelli et al. Maxima H Minus Reverse Transcriptase (200 U/µL) (2013) Smart-seq2 for sensitive full-length transcriptome profiling Introductory Vignettes. 1 ), we use the other batches to define an appropriate threshold via subset=. SMART-seq2 is an improved version of SMART-seq. 2), low-volume Smart-seq3 and Smart-seq3xpress implementations, at the indicated read depths (n = 109 We obtained a read count matrix for the SMART-Seq2 dataset (Nestorawa et al. 原理图如下：. bam" -print` mm10. 我们首先看看其建库原理。. 5) and CEL-Seq2 (ref. , 2018] to the STARmap cells, we first integrated the two STARmap replicates using the Seurat v3 integration method. We would like to show you a description here but the site won’t allow us. 将会对应课程的全部上游分析第1-4讲. We combined droplet-based encapsulation (using the 10x Genomics Chromium system) 13 and plate-based Smart-seq2 14 single-cell transcriptome profiles from the maternal–fetal interface (11 deciduas and 5 placentas from 6–14 gestational weeks) and six matched peripheral blood mononuclear cells (Fig. major cell types), or the smaller but finer cell groups are returned (e. 9. , 2016, 2018] to the STARmap cells, we first integrated the two STARmap replicates using the Seurat v3 integration method. 关注 12. In your case, you can merge all layers and split again based on batch information. Power analysis showed that Drop-seq provides a less costly option for a larger number of cells analyzed, whereas MARS-seq, SCRB-seq, and Smart-seq2 were suitable for fewer cells. SMART-seq2 (Picelli et al. 数据、软件准备. This integration task poses several challenges including donor-level This pipeline was developed to process single cell RNA-seq data generated using the Smart-Seq2 protocol. 库大小定义为每个细胞的所有相关feature的总计数之和。 Dec 11, 2022 · The library preparation of the scRNA-Seq experiments was obtained following the smart-Seq2 method [], with some of the modifications proposed in []. et al. 1 and 1, and which is the best option largely depends on the aim of the analysis. rpca) that aims to co-embed shared cell types across batches: We would like to show you a description here but the site won’t allow us. 4: New York Genome Center, New York, NY, USA, default parameters) for highly variable genes and unsupervised hierarchical clustering (Fig. gtf Dec 7, 2020 · Seurat implements the method proposed by Tirosh et al. Mar 23, 2020 · Smart-seq是由路德维格癌症研究所的 Rickard Sandberg实验室 所开发的一套在全转录组范围进行 单细胞RNA测序 (scRNA-seq) 的方法。. Switching mechanism at the 5′ end of the RNA template (SMART) protocols, such as Smart-seq2 (ref. Finally, we sought to directly compare the performance of CEL-Seq2 to another single-cell method by studying an identical cell type. Leukocyte. Jun 13, 2019 · Before transferring transcriptome-wide gene expression data from the SMART-seq2 dataset [Tasic et al. This stands in contrast to bulk RNA sequencing, which provides information only on the average transcript expression within a population of cells. The code for the new methodology is implemented in Seurat v3. The entire protocol takes ∼2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1-3 d depending on the strategy and sequencer. We also compare with Smart-Seq to demonstrate CEL-Seq2's increased sensitivity relative to other available methods. SRR文件下载到SRRfile文件下 May 25, 2021 · For both Seurat and SC3, the cell numbers at which their performance saturates are close to the cell numbers in real data: 317 cells in the Fluidigm C1 data and 1078 cells in the Smart-Seq2 data. For convenience, we distribute this dataset through our SeuratData package. This tutorial implements the major components of a standard unsupervised clustering workflow including QC and data filtration, calculation of May 12, 2023 · Actually, we don't have functions to rename layers. 1) (SS2), have shaped the single-cell transcriptomic field. For example, the median number of detected Among the latter, Smart-seq2 is still considered the “gold standard” due to its sensitivity, precision, lower cost, scalability and for being easy to set up on automated platforms. 1), including two low-throughput plate-based methods (Smart-seq2 (ref. Smart-seq2是由Picelli等人从Smart-seq中改良而来 (Picelli et al. 5. Further analysis was done within v3. 39 to score cells based on the averaged normalized expression of S. BCR analysis using BraCeR. Currently there are several different protocols in use, e. Most methods performed well for both TM lung datasets with ARI > 0. , 2016, Tasic et al. 在Smart-seq2技术基础上衍生的其它单细胞转录组测 Jul 4, 2020 · Smart-seq3既然号称能够在转录本水平研究单细胞的异质性，那么它究竟是如何达到的呢？. Smart-seq2 for sensitive full-length transcriptome profiling Compared to another scRNA-seq tool, Smart-seq2, it has a higher information loss rate for genes with lower expression levels and is less sensitive overall than full-length transcriptome sequencing Apr 18, 2020 · CRC. The Smart-seq2 Single Sample Pipeline (SS2) is designed by the Data Coordination Platform of the Human Cell Atlas to process single-cell RNAseq (scRNAseq) data generated by Smart-seq2 assays. 2015) There are also commercial platforms available, including the Fluidigm C1, Wafergen ICELL8 and the 10X Genomics Chromium; Several computational analysis methods from bulk RNA-seq can 单细胞smart-seq2建库原理. Smart-seq因为以全长mRNA建库，所以对转录本的测序覆盖度也有所上升。. May 30, 2022 · Box plots show the number of genes detected per HEK293FT cell in full-volume Smart-seq3 (ref. The integration example in the vignette used SCTransform on data from all different technologies including SmartSeq2, but the preprint paper suggests that the statistics behind SCTransform is based on negative binomial distribution of UMI. Feb 18, 2021 · As described previously, the number of detected genes for each cell obtained by 10x is significantly lower than that obtained by Smart-seq2 [30, 32]. For example, >library(Seurat) >library(SeuratData) >options(Seurat. bams/ -name "*. ) 37 under the GEO accession GSE81682, and considered 765 annotated progenitors cells expressing at least 4,000 genes. While the analytical pipelines are similar to the Seurat workflow for single-cell RNA-seq analysis, we introduce updated interaction and visualization tools, with a particular emphasis on the integration of spatial and molecular information. Although more labor intensive and expensive than emulsion droplet methods, they allow a much deeper analysis of the transcriptome, enabling the characterization at single-cell resolution of splice isoforms, allelic variants and For the purposes of this example, we’ve chosen human pancreatic islet cell datasets produced across four technologies, CelSeq (GSE81076) CelSeq2 (GSE85241), Fluidigm C1 (GSE86469), and SMART-Seq2 (E-MTAB-5061). If you run SCTResults(object=merge, slot="umi. 2012) and Drop-seq (Macosko et al. It resulted in our 2013 publication ("Smart-seq2 for sensitive full-length transcriptome profiling in single cells" by Picelli, S; Björklund, ÅK; Faridani, OR; Sagasser, S; Winberg, G; Sandberg, R. integrated. Seurat is an R package designed for QC, analysis, and exploration of single-cell RNA-seq data. Suggesting can't use the {"payload":{"allShortcutsEnabled":false,"fileTree":{"section02-scRNA":{"items":[{"name":"figures","path":"section02-scRNA/figures","contentType":"directory"},{"name The droplet-based 10X Genomics Chromium (10X) approach and the plate-based Smart-seq2 full-length method are two frequently used scRNA-seq platforms, yet there are only a few thorough and systematic comparisons of their advantages and limitations. The pipeline's workflow is written in WDL, is freely available on GitHub, and can be run by any compliant WDL runner (e. , 2014). gz Data processing The Smart-seq2 data were first processed to filter out low-quality reads when: (1) “N” bases accounting for 10% read length; or (2) bases with quality < 5 account for 50% read length; or (3) containing adapter sequences. In addition, as we do not have UMI counts, we use lognormalization rather than SCT. The method currently supports five integration methods. / data/e12. May 8, 2022 · Smart-seq2上游处理（Hisat2版） 最近试了很多Smart-seq2上游处理的方法，参照了Jimmy老师的教程，以及阿则的生信小站公众号文章。对代码进行了整理。但是过程中仍然有一些问题尚待解决。 1. First, we identified anchors between the STARmap datasets using the FindIntegrationAnchors function in Apr 6, 2020 · Here, we systematically and directly compared seven methods (Fig. assay") just before running PrepSCTFindMarkers, you may notice that one of your layers has "Spatial" while the others have "RNA" listed. The metadata file contains the technology (tech column) and cell type annotations (cell type column) for each cell in the four datasets. The recommended processing step is then to use velocyto. The droplet-based 10X Genomics Chromium (10X) approach and the plate-based Smart-seq2 full-length method are two frequently used scRNA-seq platforms, yet there are only a few thorough and systematic comparisons of their advantages and limitations. 从上面的建库原理图来看，其建库方式基本上与smart-seq2一致，最大的不同在于在Tn5酶的tag后面添加了UMI序列（上图中的红色x号 Here, we introduce Smart-seq2 with improved reverse transcription, template switching and preamplification to increase both yield and length of cDNA libraries generated from individual cells. Jan 19, 2022 · For example, a study demonstrated that Smart-seq2 can detect a larger number of expressed genes than other scRNA-seq protocols such as CEL-seq2, MARS-Seq, Smart-seq, and Drop-seq , while MATQ-seq, another full-length transcript sequencing platforms, outperforms Smart-seq2 in detecting low-abundance genes. 6)) and five Apr 11, 2022 · Hello I am new to RNA seq and ScRNA-seq i have smart-seq2 data want to convert to gene level TPM for this I need gene length info how to get this information I tried to get the complete length of gene by substracting gene START and END but. py command line tool to annotate spliced, unspliced and spanning reads in the measured cells: velocyto run_smartseq2 -d 1 `find . Seq2 Drop-seq inDrops Seq-Well ## 526 6584 6584 3773 ## Smart-seq2 ## 526 . May 4, 2020 · Smart-seq2 cDNA libraries were generated according to the published protocol 22. We used Salmon7 to obtain isoform expression levels, and we performed clustering and cell-type identification ( Supplementary Fig. 应用单细胞测序技术研究科学问题越来越普遍，当下应用最火热的是10X Genomics公司的解决方案，但是在某些特殊或者少量细胞样本的单细胞转录组研究中，Smart-seq2技术还是一项研究利器。. Bioanalyzer (Agilent) was used to assess the quality of libraries, and the good-quality ones were then quantified with KAPA Library Quantification Kit (Roche), and finally submitted for sequencing. The goal of integration is to ensure that the cell types of one condition/dataset align with the same celltypes of the other conditions/datasets (e. (2) Include methyl group donor betaine in Oct 29, 2019 · （二）tSNE分析（Seurat方法） 在分析之前，先简单的了解一下什么是Seurat，这是这个包的官方网站（ SATIJA LAB），据说这个包今年刚升过级，这个包还可以很友好的直接读取10x Genomics的输出结果（单细胞转录组测序分析--初探Seurat）。有几个比较好的文章对于这个 SMART-seq2 is the improved version of SMART-seq (similar to Fluidigm C1), featuring refinements to the reverse transcription, template switching, and pre-amplification steps in order to increase yield and length of cDNA libraries generated from each individual cell (while also using off-the-shelf reagents that are available at lower cost Mar 30, 2022 · Other approaches such as SMART-seq2 and CEL-seq2 can be used to profile cells that have been sorted While packages such Seurat or Scanpy offer the ability to calculate some basic QC metrics Oct 31, 2023 · Seurat v5 enables streamlined integrative analysis using the IntegrateLayers function. cell subtypes). txt. About Seurat. 2万. . 0 relies on anchor cells between pairs of datasets, which are Dec 24, 2020 · As shown in Table 2, for two TM full datasets from 10X and Smart-Seq2 platforms (with 32 and 37 cell types, respectively), we used a subset to create two smaller TM lung datasets (with 8 and 10 cell types, respectively). 3. The commonly used resolution ranges between 0. smartseq2得到的文件和10X的不一样，10X的数据虽然也有R1、R2两个数据，但第一个存储的是barcode和UMI信息，而只有第二个才是真正的测序信息，也就是单端测序；而 Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. object. Apr 28, 2016 · We implemented CEL-Seq2 on Fluidigm's C1 system, providing its first single-cell, on-chip barcoding method, and we detected gene expression changes accompanying the progression through the cell cycle in mouse fibroblast cells. The metadata contains the technology ( tech column) and Nov 11, 2018 · Maternal and fetal cells in early pregnancy. The UMI strategies encompass two plate-based (SCRB-seq, CEL-seq2) and the most common non-commercial and Apr 17, 2020 · For the purposes of this example, we’ve chosen human pancreatic islet cell datasets produced across four technologies, CelSeq (GSE81076) CelSeq2 (GSE85241), Fluidigm C1 (GSE86469), and SMART-Seq2 (E-MTAB-5061). With recent technological advancements and decreasing sequencing costs, scRNA-seq has become Smart-seq2 was developed in 2012 in the Sandberg lab in order to improve on the Smart-seq while reducing the cost. Each of these methods performs integration in low-dimensional space, and returns a dimensional reduction (i. This protocol presents a plate-based workflow to perform RNA sequencing analysis of single cells/nuclei using Smart-seq2. The current We would like to show you a description here but the site won’t allow us. S7 ). g. 充电. Smart-seq2 transcriptome libraries have improved detection, coverage, bias and accuracy compared to Smart-seq libraries and are generated with off-the Single-cell RNA-seq (Smart-SEQ2) of innate lymphoid cells from Head and Neck Squamous Cell Carcinoma - josh-tay/HNSCC_NK_scRNA Oct 23, 2019 · 上游分析. Crowmell ). The table Aug 6, 2020 · Summary. Hi, I am certainly not on the official Seurat team, but I had a similar issue and I can tell you how I got around it. Oct 11, 2019 · Here, we included one full-length method (Smart-seq2 13) and four UMI methods 2,14,15,16. The cDNA libraries were used for short-read (Illumina Hiseq) or long-read (Nanopore or Jul 2, 2020 · Yet Smart-seq2 lacks a unique molecular identifier (UMI), valuable for profiling isoform-level RNA counting. Mar 2, 2021 · Single-cell RNA sequencing (scRNA-seq) is generally used for profiling transcriptome of individual cells. assay. While they are technically performed in the same lab using similar technology, we see clear batch effects in the data if we do not integrate. Data preprocessing. First, we identified anchors between the STARmap datasets using the FindIntegrationAnchors function in Seurat v3, using Seurat (anchors and CCA) First we will use the data integration method presented in Comprehensive Integration of Single Cell Data. In addition, scRNA-seq protocols vary . For the purposes of this example, we’ve chosen human pancreatic islet cell datasets produced across four technologies, CelSeq (GSE81076) CelSeq2 (GSE85241), Fluidigm C1 (GSE86469), and SMART-Seq2 (E-MTAB-5061). We therefore performed CEL-Seq2 on mouse dendritic cells – formerly the subject of intense analysis using Smart-Seq on the C1 . The entire protocol takes ∼2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1–3 d depending on the strategy and sequencer. The pipeline features: Quality control using fastqc and multiqc; Alignment using STAR; Quantification using rsem or featureCounts; TCR analysis using TraCeR. May 30, 2022 · Main. 1 and Extended Data Fig. control Contribute to jmzeng1314/scRNA_smart_seq2 development by creating an account on GitHub. Oct 31, 2023 · Seurat v5 enables streamlined integrative analysis using the IntegrateLayers function. 5b). e. Here, by directly comparing the scRNA-seq data generated by these two platforms from the same Hello, I would like to continue a topic that was first started on this Biostars post. 1: Distribution of each QC metric across cells from each donor of the Segerstolpe pancreas dataset. Nov 14, 2020 · 我们使用几种常见的QC指标，根据表达谱来鉴定低质量的细胞。这些指标将在下面针对SMART-seq2数据的reads进行质控，但相同的过程适用于由其他技术（如MARS-seq和基于液滴的方法）生成的UMI数据。 1. In this chapter I describe how to efficiently generate sequencing-ready libraries, highlight common issues and pitfalls, and offer solutions for generating high We assume that the SMART-seq2 data has been demultiplexed and aligned, so that we have one bam file per cell. Load in expression matrix and metadata. Aug 18, 2019 · 单细胞分析seurat包学习笔记2 不同数据集联合分析. Seurat aims to enable users to identify and interpret sources of heterogeneity from single-cell transcriptomic measurements, and to integrate diverse types of single-cell data. For new users of Seurat, we suggest starting with a guided walk through of a dataset of 2,700 Peripheral Blood Mononuclear Cells (PBMCs) made publicly available by 10X Genomics. Figure 8. We describe (1) the dissociation procedures for cell/nucleus isolation from the mouse brain and human organoids, (2) the flow sorting of single cells/nuclei into 384-well plates, and (3) the preparation of libraries following miniaturization of the Smart-seq2 Front Page | Statistical Science For some batches that seem to have a majority of low-quality cells (Figure 8. Then layer names will be meaningful. However, SMARTseq2 is Nov 18, 2023 · 47 stored in SRA usually contain read1, read2 and index reads, while those of Smart-seq2 48 (Picelli et al. In conclusion, we use scDesign2 to find that the cell numbers are close to being optimal in the CEL-Seq2, Fluidigm C1, and Smart-Seq2 datasets. Essentially, in an attempt to help the OP from that topic, I brought the point of downstream data analysis with full-length RNA-seq protocol such as Smart-Seq2 when one uses Salmon (in quasi-mapping mode) and tximport. describe Smart-seq3, which further improves the sensitivity of Apr 28, 2016 · While Smart-Seq2 has an improved template switch step relative to Smart-Seq , previous work has also shown that C1-based transcriptomics shows increased sensitivity , thus making for an appropriate comparison. However, after iMAP correction, we recovered the dropout genes by 10x and boosted the number to be almost at the same level as Smart-seq2 (Fig. Each point represents a cell and is colored according to whether that cell was May 6, 2019 · Seurat CCA was unsuccessful at integrating these (the MARS-Seq dataset has a relatively low average percentage of nonzero genes at 30% versus the Smart-seq2 dataset with an average of 79% Mar 3, 2020 · Examples include Seurat 6,13,21, pcaReduce 22, MNN batch-effect-correction 23, RaceID3 24, The FACS-sorted SMART-seq2 dataset contained count data for 23,433 genes across 53,760 cells, with an Oct 31, 2023 · This tutorial demonstrates how to use Seurat (>=3. The protocols of SMART-seq and SMART-seq2 are almost the same. The multiple types of Seurat Scanpy Reagents * Antibodies. rpca) that aims to co-embed shared cell types across batches: Here, the resolution parameter is used to control whether the major and coarsed cell groups (e. 1 of Seurat 25 retaining cells with more than 500 genes detected (intron Apr 1, 2021 · With the improvement of sequencing technologies, scRNA-seq is becoming robust and accessible for transcriptome analysis. Two key parameters are: (1) exchanging the last guanylate for a locked nucleic acid (LNA) at the 3' end of TSO. Metadata. Mar 1, 2022 · Due to experimental considerations, the analysis of transcript variation in 10x Chromium data is mostly restricted to the 3’ end of genes; in contrast, Smart-seq2 and other full-length, short-read protocols theoretically enable characterization of transcript variation along the whole gene. For convienence, we distribute this dataset through our SeuratData package. (2018) ]. 2013), CELL-seq (Hashimshony et al. , 2013) (Picelli Integration is a powerful method that uses these shared sources of greatest variation to identify shared subpopulations across conditions or datasets [ Stuart and Bulter et al. 2) to analyze spatially-resolved RNA-seq data. version = 'v5') >obj <- LoadData(ds = 'pbmcsca') Oct 27, 2023 · Single-cell RNA sequencing (scRNA-seq) is a method used to study gene expression at the single-cell level. 这部分和转录组分析很相似，所以也是简单带过. Mar 2, 2021 · Abstract. 29 were sequenced using smart-seq2, all other batches were sequenced using 10X Genomics protocol. Protein. 「生信技能树」使用Smart-seq2单细胞转录组数据探索小鼠性腺发育共计14条视频，包括：01-服务器数据处理环境配置、02-下载数据并且比对、03-转录组定量等，UP主更多精彩视频，请关注UP账号。. Nov 27, 2023 · While the cells in ref. genes. Sep 27, 2019 · To explore the homogeneity and heterogeneity of myeloma among patients, 126 BM cells were analyzed by seurat (version 2. Basic QC and normalization has been performed, as described earlier in this workshop. Hagemann-Jensen et al. Single-cell RNA sequencing (scRNA-seq) is generally used for profiling transcriptome of individual cells. We will add this functionality soon. Feb 28, 2024 · Seurat V5 has gradually gained popularity due to its faster running speed. 1a, b, Supplementary Tables 1, 2, Extended Data Apr 28, 2016 · CEL-Seq2 shows better sensitivity and reproducibility than Smart-Seq. It is important to note that CEL-Seq2, as a 3′ tag method, differs from Smart-Seq, which produces full-length transcripts. Smart-seq2 transcriptome libraries have improved detection, coverage, bias and accuracy compared to Smart-seq libraries and are generated with off-the The code below downloads a Seurat object that contains human pancreatic islet cell data from four single cell sequencing technologies, CelSeq (GSE81076), CelSeq2 (GSE85241), Fluidigm C1 (GSE86469), and SMART-Seq2 (E-MTAB-5061). May 11, 2020 · We combined four publicly available datasets on human pancreas generated using Fluidigm C1 14, SMART-seq2 15, Seurat 3. Single-cell gene expression analyses hold promise for characterizing Mar 27, 2023 · For the purposes of this example, we’ve chosen human pancreatic islet cell datasets produced across four technologies, CelSeq (GSE81076) CelSeq2 (GSE85241), Fluidigm C1 (GSE86469), and SMART-Seq2 (E-MTAB-5061). Smart-seq2 is based on microtiter plates [10], [11], where mRNA is separated and Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. 选择了通过四种技术产生的人胰岛细胞数据集：CelSeq（GSE81076) CelSeq2（GSE85241），Fluidigm C1（GSE86469）和SMART-Seq2（E-MTAB-5061）。我们在此处提供组合的原始数据矩阵和相关的元数据文件以便开始。 数据集预处理 Jun 6, 2019 · Before transferring transcriptome-wide gene expression data from the SMART-seq2 dataset [Tasic et al. Since Here, we introduce Smart-seq2 with improved reverse transcription, template switching and preamplification to increase both yield and length of cDNA libraries generated from individual cells. May 10, 2022 · We are working to integrate two smart-seq2 based data-sets together. 2014) datasets only include read1 and read2; the count matrix output by 49 CellRanger consists of barcode, matrix and feature files, while other preprocessing 50 tools usually output a single count matrix file. Overall, SMARTseq2 was found to be the most sensitive and accurate method to analyze full-length transcriptomes and detect alternative splice forms . I was going through many biostar threads gene length for calculating TPM values. May 31, 2023 · A systematic comparison of some scRNA-seq technologies including SMART-seq, SMART-seq2, CEL-seq2, SCRB-seq, and MARS-seq was performed using mouse embryonic stem cells . Sep 22, 2013 · Nature (2023) Smart-seq2 improves yield and length in single cell-derived cDNA libraries and uses off-the-shelf reagents. 5A). 其实在Seurat v3官方网站的Vignettes中就曾见过该算法，但并没有太多关注，直到看了北大张泽民团队在2019年10月31日发表于Cell的《Landscap and Dynamics of Single Immune Cells in Hepatocellular Carcinoma》，为了同时整合两类数据（包括SMART-seq2和10X）（Hemberg-lab单细胞转录组 生信技能树 伴你共成长. ) in Nature methods. The authors generously provided lineage annotations for each cell (corresponding to Figure 4 in the original publication, used in our Figure 3 ). ha rl my dc ws sv vu br hu vo