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Sample GSM4766480 Query DataSets for GSM4766480
Status Public on Oct 02, 2020
Title 10x mRNA Single cell sequencing of IELs 2
Sample type SRA
Source name Intraepithelial lymphocytes (IELs)
Organism Mus musculus
Characteristics strain: Foxp3eGFP-CreERT2 Rosa26|s|-td-Tomato
tomato: NEG
sequencing batch: Seq.10x.batch1
experiment batch: Exp.10x.batch1
cell: CD4 cells from intestinal epithelium
Extracted molecule total RNA
Extraction protocol Tomato+ and Tomato- CD4+ IELs were sorted, counted for viability and immediately subjected to library preparation. The scRNA-seq and scTCR-seq libraries were prepared using the 10x Single Cell Immune Profiling Solution Kit, according to the manufacturer’s instructions at the Genomics core of Rockefeller University.
 The scRNA libraries were sequenced on an Illumina NextSeq500 to a minimum sequencing depth of 50,000 reads per cell using read lengths of 26 bp read 1, 8 bp i7 index, 98 bp read 2. 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description RNA.Meta.csv
Data processing Raw fastq files derived from our RNA-seq libraries were processed with cellranger count (v3.1.0) using the 10x Genomics prebuilt mouse reference (v3.0.0 mm10). Our libraries were processed independently and merged into a single experiment at the analysis level using Seurat (v3.1.1) (Stuart et al., 2019). Quality control was performed by removing cells with high (> 5%) mitochondrial UMI content. Cells having more than 4000 or less than 200 genes were excluded from our analysis. TCR contigs and annotation were performed with the Cellranger vdj workflow from 10x Genomics and the prebuild mouse reference (v3.1.0 mm10). Paired TCR clones were defined by the V, (D), J and CDR3 nucleotide composition for alpha and beta chains. TCR clone sharing was assigned to cells expressing identical V, (D), J, and CDR3 nucleotide and amino acid sequences.  
Raw fastq files were processed by using the mouse transcriptome (gencode M23) with the kallisto (v0.46) software (Bray et al., 2016). Analysis of transcript quantification was performed at the gene level by using the sleuth (v0.30) package for R (Pimentel et al., 2017). Shortly, we modeled batch effect and our experimental design using the sleuth_fit function and detected differentially expressed genes between all groups by the likelihood ratio test (lrt). To spot significantly expressed genes between group pairs, we used the wald-test function. Genes with false discovery rate less than 0.05 and 1 log2 fold-change were used in downstream analysis. Gene set enrichment analysis (GSEA) was performed by using signature gene sets in gmt format and a pre-ranked gene list by log2 fold-change between two groups as input for the fgsea package/R (Korotkevich et al., 2019). Gene ontology (GO) analysis was executed by comparing all detected genes as our background and the lists of differentially expressed genes against the biological processes gene sets by using topGO/R (Alexa and Rahnenfuhrer, 2019; Carlson M, 2019). 
For Miseq data, fastq files were de-multiplexed and paired-end reads were assembled at their overlapping region using PANDASEQ (Masella et al., 2012) and FASTAX toolkit. Demultiplexed and collapsed reads were assigned to wells according to barcodes. Fasta files from both Sanger and Miseq sequences were aligned and analyzed on IMGT ( (Brochet et al., 2008). Cells with identical TCRb CDR3 nucleotide sequences were considered as the same clones. Clonality was confirmed by sequencing TCRa of the expanded clones as assessed by TCRb sequencing for mice shown in Figure S2F and H. Clonality was assigned based on paired TCRab for mice shown in Figure 2 and S2C. Only in frame junction sequences of TCRb were included in the analysis. For fixed-Vb6 mice shown in Figure 2 and S2, each TCRa CDR3 was considered a clone.
Genome_build: mm10
Submission date Sep 03, 2020
Last update date Oct 03, 2020
Contact name Tiago Castro
Organization name The Rockefeller University
Lab Laboratory of Mucosal Immunology
Street address 1230 York Avenue
City New York
State/province New York
ZIP/Postal code 10065
Country USA
Platform ID GPL19057
Series (1)
GSE157453 T cell receptor is required for differentiation but not maintenance of intestinal CD4+ intraepithelial lymphocytes
BioSample SAMN16055606
SRA SRX9072456

Supplementary file Size Download File type/resource
GSM4766480_Velocity_Tomato_Pos.loom.gz 10.3 Mb (ftp)(http) LOOM
GSM4766480_barcodes.pos.tsv.gz 5.8 Kb (ftp)(http) TSV
GSM4766480_features.pos.tsv.gz 272.8 Kb (ftp)(http) TSV
GSM4766480_matrix.pos.mtx.gz 6.3 Mb (ftp)(http) MTX
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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