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| Status |
Public on Oct 02, 2020 |
| Title |
Single cell TCR sequencing of CD4+ Tcells from the intestinal epithelium 5 |
| Sample type |
SRA |
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| Source name |
Intraepithelial lymphocytes (IELs)
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| Organism |
Mus musculus |
| Characteristics |
strain: Foxp3eGFP-CreERT2 Rosa26|s|-td-Tomato
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single cells were index-sorted using a FACS Aria into 96-well plates containing 5μL of lysis buffer (TCL buffer, Qiagen 1031576) supplemented with 1% β-mercaptoethanol) and frozen in -80°C prior to RT-PCR.
RNA and RT-PCRs for TCRa and TCRb were prepared as previously described (Dash et al., 2011). PCR products for TCRa and TCRb were either subjected to Sanger sequencing or multiplexed with barcodes and submitted to MiSeq sequencing (Han et al., 2014) using True Seq Nano kit (Illumina).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Library strategy: Amplicon-seq
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 (imgt.org/HighV-QUEST) (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
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| Submission date |
Sep 03, 2020 |
| Last update date |
Oct 03, 2020 |
| Contact name |
Tiago Castro |
| E-mail(s) |
trezende@rockefeller.edu
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| Organization name |
The Rockefeller University
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| Lab |
Laboratory of Mucosal Immunology
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| Street address |
1230 York Avenue
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL16417 |
| Series (1) |
| GSE157453 |
T cell receptor is required for differentiation but not maintenance of intestinal CD4+ intraepithelial lymphocytes |
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| Relations |
| BioSample |
SAMN16055583 |
| SRA |
SRX9072481 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4766505_200515AB.fasta.gz |
89.4 Kb |
(ftp)(http) |
FASTA |
| GSM4766505_200515AB.xlsx |
296.9 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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