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Sample GSM3082689

Query DataSets for GSM3082689

Status

Public on Sep 25, 2018

Title

Treg_ATAC_Colon_Nrp1p_1

Sample type

SRA

Source name

Colonic lamina propria

Organism

Mus musculus

Characteristics

mouse id: 5
strain: C57BL/6
tissue: colon
cell type: CD4+CD25+Treg

Extracted molecule

genomic DNA

Extraction protocol

10,000 cells were sorted into collection buffer (PBS, 0.5% BSA, 2mM EDTA), pelleted and lysed in hypotonic buffer (10mM Tris-HCl (pH 7.5), 10mM NaCl, 3mM MgCl2, 0.1% NP-40). Nuclei were pelleted by centrifugation for 30 min. at 500g, 4oC.
ATAC-seq adapted from Buenrostro et al., 2013 and Lara-Astiaso et al., 2014. Nuclear pellets were re-suspended in 5uL of transposition mix (1uL Tagment DNA Enzyme, 2.5uL Tagment DNA Buffer from Nextera DNA Sample Prep Kit, 1.5uL H2O), and incubated at 37C for 60min. Two sequential PCR reactions were performed. After the first PCR, libraries were selected for small fragments (< 600bp) using SPRI beads followed by a second round of PCR with the same conditions, and a final SPRI bead purification to capture all fragments for the final library.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

Cln_Treg_merged_rep1.bw

Data processing

Base calling was performed with Real-Time Analysis v2.4.11
ATAC-seq reads from two replicates per sample were filtered for quality using sickle v1.2 (default settings for PE reads), adapter-trimmed using cutadapt v1.8.3 (`-e 0.1, -m 20`), and mapped to mm9 using Bowtie v2.2.4, keeping only mate pairs that mapped to a single best location using samtools v1.3, and were non-duplicates using Picard v1.130.
To generate bigWig files, filtered and mapped reads were used to generate tag directories in HOMER v4.6 (`makeTagDirectory -tbp 1 -checkGC -illuminaPE`), followed by bedGraph files (HOMER `makeUCSCfile -fsize 1e50 -norm 1e6`), which were then used to generate bigWig files using UCSC-tools (`bedGraphToBigWig`). For colon Treg samples, raw data files were generated from purified sub-populations of Nrp1(+) and Nrp1(-) Tregs. For each replicate, a combined Treg sample was derived by randomly sampling from Nrp1+ and Nrp1- .bam files and combining reads in the proportions in which these Tregs are found in colonic lamina propria (~50% Nrp1+/50% Nrp1- for the age of mice used in this study). This combined .bam file was then used to generate a processed bigWig file in the same pipeline outlined above. This process was repeated for splenic Treg samples corresponding to the Nrp1(+) and Nrp1(-) populations, except this time the proportions in the final file were 90% Nrp1(+), 10% Nrp1(-), based on flow-cytometric analysis of splenic CD4+FOXP3+ Tregs from the mice used in our study.
The data in the BED file represent the mm9 chromosomal coordinates of the complete set of 75,363 Treg open chromatin regions (OCRs) obtained by merging the individual peak/OCR files from all Treg ATAC-seq samples, as detailed in the methods of the manuscript DiSpirito, Zemmour et al. (pending).
Genome_build: mm9
Supplementary_files_format_and_content: bigWig, BED

Submission date

Apr 05, 2018

Last update date

Sep 25, 2018

Contact name

CBDM Lab

E-mail(s)

cbdm@hms.harvard.edu

Phone

617-432-7747

Organization name

Harvard Medical School