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Series GSE146292 Query DataSets for GSE146292
Status Public on Jan 04, 2021
Title Stepwise chromatin and transcriptional acquisition of an intraepithelial lymphocyte program
Organism Mus musculus
Experiment type Expression profiling by high throughput sequencing
Genome binding/occupancy profiling by high throughput sequencing
Summary Mesenteric lymph node (mLN) T cells undergo tissue adaptation upon migrating to intestinal lamina propria (LP) and intraepithelial (IE) compartments, ensuring appropriate balance between tolerance and resistance. By combining mouse genetics with single-cell and chromatin analyses, we addressed the molecular imprinting of gut epithelium on T cells. Transcriptionally, conventional and regulatory (Treg) CD4+ T cells from mLN, LP and IE segregate based on the gut layer they occupy; trajectory analysis suggests a stepwise loss of CD4-programming and acquisition of an intraepithelial profile. Treg fate–mapping coupled with RNA– and ATAC–sequencing revealed that the Treg program shuts down before an intraepithelial program becomes fully accessible at the epithelium. Ablation of CD4 lineage–defining transcription factor ThPOK results in premature acquisition of an IEL profile by mLN Tregs, partially recapitulating epithelium imprinting. Thus, coordinated replacement of circulating lymphocyte program with site–specific transcriptional and chromatin changes is necessary for tissue imprinting.
Overall design Atac-Seq: ATAC-Seq was performed on 5,000-40,000 FACS-purified cells from 2-9 mice. In brief, cells were lysed in lysis buffer for 1 minute and transposed with Tagment DNA Enzyme 1 (Illumina) for 30 minutes. DNA was cleaned up using a MinElute DNA purification Kit (Qiagen), followed by barcoding and library preparation by the Nextera DNA Library preparation kit (Illumina) according to manufacturer’s guidelines and sequenced on an Illumina Illumina NextSeq 500.

ChIP-Seq: Cells were fixed in 1% formaldehyde for 20min, quenched with 0.15M glycine and washed in PBS. 2x106 were then sorted and lysed for 30min at 4ºC. Cells were then sonicated for 22 minutes at 30 seconds on/off using the Bioruptor sonicator (Diagenode) and spun down. 10% was frozen for input, the remaining 90% was incubated with anti-ThPOK antibody bound to goat anti-rabbit M280 magnetic beads (Invitrogen) overnight at 4ºC prior to washing with RIPA buffer and overnight decrosslinking at 65ºC. DNA was then eluted off beads into TE buffer and, along with input, purified using the Zymogen DNA clean & concentrator kit. DNA was sequenced using NextSeq2500. To obtain Tregs, Foxp3RFP reporter mice were injected with IL-2/aIL-2 complex as previously described for 3 consecutive days for Treg expansion. CD4+ T cells were isolated from the spleen and mLNs and sorted as Thy1+TCRb+CD4+CD8a–RFP+.

RNA-Seq: Sorted cells (300-800) were lysed in TCL buffer (Qiagen, 1031576) supplemented with 1% βmercaptoethanol. RNA was isolated using RNAClean XP beads (Agentcourt, A63987), reversibly transcribed, and amplified. Uniquely barcoded libraries were prepared using Nextera XT kit (Illumina) following manufacturer’s instructions. Sequencing was performed on an Illumina NextSeq 550.

Single Cell Sequencing: Lymphocytes were sorted, counted for viability and immediately subjected to library preparation. The scRNA-Seq library was prepared using the 10x Single Cell Chromium system, according to the manufacturer’s instructions at the Genomics core of Rockefeller University and was sequenced on an Illumina NextSeq 550 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.

Please note that the processed data generated from both replicates is linked to the corresponding rep1 sample (with the lower GSM accn number).
Contributor(s) London M, Bilate A, Castro TB, Mucida D
Citation(s) 33686285
Submission date Mar 03, 2020
Last update date Apr 05, 2021
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
Platforms (3)
GPL17021 Illumina HiSeq 2500 (Mus musculus)
GPL19057 Illumina NextSeq 500 (Mus musculus)
GPL21626 NextSeq 550 (Mus musculus)
Samples (40)
GSM4376809 Sample 1_ATAC_exTreg-IEL
GSM4376810 Sample 2_ATAC_exTreg-IEL
GSM4376811 Sample 3_ATAC_CD4-IEL
BioProject PRJNA610060
SRA SRP251581

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE146292_RAW.tar 4.3 Gb (http)(custom) TAR (of BB, BED, BIGWIG, H5, MTX, NARROWPEAK, TSV)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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