DOI: 10.5061/dryad.gtht76hv9
title: Transcription start site analysis for heterogenous CD4+ T cells using 5′ scRNA-seq

description: These datasets are generated by ReapTEC (read-level pre-filtering and
 transcribed enhancer call) using 5' single-cell RNA-seq data on human
 heterogenous CD4+ T cells. By taking advantage of a unique “cap signature”
 derived from the 5′-end of a transcript, ReapTEC simultaneously profiles
 gene expression and enhancer activity at nucleotide resolution using
 5′-end single-cell RNA-sequencing (5′ scRNA-seq). The detail of ReapTEC
 pipeline is described in https://github.com/MurakawaLab/ReapTEC.
description: README: Transcription start site analysis for heterogenous CD4+ T cells
 using 5′ scRNA-seq https://doi.org/10.5061/dryad.gtht76hv9 **Description
 of the data and file structure** **Data_summary.xlsx.zip: Summary of
 single-cell experiments in this study.** **5scCTSSbed_All.zip**: There are
 102 files containing count data for analyzing transcription start site
 (TSS) signals. Details are as follows. Our original raw sequencing data
 and processed data of 5′ scRNA-seq have been deposited to National
 Bioscience Database Center (NBDC) Human Database (accession code:
 hum0350). Raw sequencing data originated from human subjects have been
 deposited to Japanese Genotype-phenotype Archive (JGA, accession code:
 JGAS000689). We retrieved 5′ scRNA-seq data for human memory CD4+ T cells
 stimulated with viral antigens from the Gene Expression Omnibus database
 (accession number GSE152522). In total, 102 5′ scRNA-seq datasets were
 processed by ReapTEC pipeline (https://github.com/MurakawaLab/ReapTEC).
 During the process, count files were generated for each TSS (CTSS files).
 The CTSS files in 5scCTSSbed_All.zip are CTSS files obtained from ReapTEC
 pipeline and further sorted by “LANG=C sort -k 4”. **Naming convention
 (Please refer to Data_summary.xlsx)** Abbreviation: D1, donor 1; D2, donor
 2; D3: donor 3 addExp_Bulk_5sc: Data originated from 5′ scRNA-seq of bulk
 CD4+ T cell corresponding to the samples written in
 "5sc_snRNA_seq_added_samples" window of the Data_summary.xlsx.
 Bulk_5sc_[replication number]: Data originated from 5′ scRNA-seq of bulk
 CD4+ T cells corresponding to the samples written in
 "5sc_3scRNA-seq_CD4bulkCITE-seq" window of the
 Data_summary.xlsx. Treg, Th1or2, Tfh, Th17, LAG3_5sc_[replication number]:
 Data originated from 5′ scRNA-seq of subpopulations corresponding to the
 samples written in "5sc_3scRNA-seq_CD4bulkCITE-seq" window of
 the Data_summary.xlsx. Treg, Th1or2, Tfh, Th17, LAG3_CITE_[replication
 number]: Data originated from 5′ CITE-seq of subpopulations corresponding
 to the samples written in " 5CITE-seq_added_samples" window of
 the Data_summary.xlsx. SoftclipG_aCD4_5sc_48h_sorted.CTSS.bed: Data
 originated from 5′ scRNA-seq of activated CD4+ T cell corresponding to the
 samples written in "5sc_snRNA_seq_added_samples" window of the
 Data_summary.xlsx. SoftclipG_[SRR number]_[stimulation hours]: Data
 originated from 5′ scRNA-seq for human memory CD4+ T cells stimulated with
 viral antigens from the Gene Expression Omnibus database (accession number
 GSE152522). **md5sum_CTSS_files.txt**: A text file containing the md5sum
 of the files included in 5scCTSSbed_All.zip.
 **20230928_TC_anno_Gene_rank_bed6.bed**: This is a bed6 file for robust
 TSS peaks generated using ReapTEC (with a cutoff of log2CPM ≥ 2 in at
 least one cell cluster across 136 cell clusters of CD4+ T cells in this
 study). Robust TSS peaks were named according to the nearest known
 transcript and numbered in ascending order from upstream to downstream of
 the transcript.
 **CD4bulk_5sc_TCver_TCRremoved.removeMono.harmonyintegration_FigS14.rds**:
 This is a Seurat object used in Supplementary figure S14 (Oguchi et al.
 Science). The sorted CTSS files in 5scCTSSbed_All.zip and
 20230928_TC_anno_Gene_rank_bed6.bed were used to count the reads mapping
 to each robust TSS peak at single cell level and create count matrix files
 for Seurat version 5 (4.9.9.9067). Of the matrix files, those for bulk
 CD4+ T cells (22 files) were processed in Seurat. Robust TSS peaks
 expressed in three or more cells were retained. Doublets were predicted
 and filtered using R package scDblFinder version 1.14.0. Singlets were
 retained for downstream analysis. Cells expressing fewer than 200 TSS
 peaks and those with more than 3% of all transcripts derived from the
 mitochondrial genome were excluded. TSS peaks of T cell receptor–related
 transcripts were excluded in this analysis. Data integration and batch
 collection were performed using Harmony version 1.0.3 in the
 “IntegrateLayers” function implemented in Seurat. Code/Software
 https://github.com/MurakawaLab/ReapTEC