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Sample GSM3938202 Query DataSets for GSM3938202
Status Public on Mar 29, 2020
Title ATAC.TCell.Donor.C.CSF2neg.1
Sample type SRA
Source name primary T lymphocytes derived from human peripheral blood mononuclear cells (PBMC)
Organism Homo sapiens
Characteristics cell type: Effector memory T lymphocytes
label: Peneg
antibody: none
Treatment protocol PMA and ionomycin stimulation for 3h for cytokine secretion assay-based separation
Extracted molecule genomic DNA
Extraction protocol Human peripheral blood mononuclear cells were separated from peripheral blood using Ficoll-Paque Plus (GE Healthcare). CD4+ T lymphocytes were further isolated by positive selection using magnetic microbeads (Miltenyi Biotec). Effector Memory T cell subsets were then sorted based on the expression of the following surface markers: CD4+CD25–CD45RA-CCR7-. TEM cells were then PE-labelled for GM-CSF expression with a GM-CSF Cytokine Secretion Assay (Miltenyi Biotec) and sorted for PE+/-.
Tagmented DNA from GM-CSF- TEM lysed with Buffer1.
50000 cells were pelleted by centrifugation and re-suspended in 50 µl of cold lysis buffer 1 (10mM Tris-HCl pH 7.4, 10mM MgCl2, 0.1% Igepal CA-630) or 2 (10mM Tris-HCl pH 7.4, 3mM MgCl2, 10mM NaCl, 0.1% Igepal CA-630). After incubation on ice for three minutes, nuclei were pelleted by centrifugation for 20 min at 500g, 4 ºC. The supernatant was discarded and nuclei were re-suspended in 50 µl of reaction buffer containing 1 µl of Tn5 transposase (made in house), 10 ul of 5x transposase buffer (50mM Tris-HCl, pH 8.4 and 25mM MgCl2), and 39uL of milliQ H2O. The reaction was incubated at 37ºC for 60 minutes with 600 RPM mixing. Then 10 µl of clean up buffer (900mM NaCl, 300mM EDTA), 4ul of 5% SDS and 2 µl of Proteinase K (20 µg/µl) (New England Biolabs) were added and incubated for 30 min at 40 ºC. Tagmented DNA was isolated using SPRI beads (2x) and amplified by PCR. Fragments smaller than 600 bp were isolated using SPRI cleanup and libraries where sequenced on an Illumina NextSeq500 platform.
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
Description ATAC.TCell.Donor.C.CSF2neg.narrowPeak
Data processing Reads were pre-processing (filtering, quality trimming and adapter clipping) using Trimmomatic (version 0.38) (PMID:24695404), then single-end with min 36-bp reads length were aligned to human genome build GRCh38/hg38 using Bowtie2 v2.2.6 (PMID: 22388286) with “–very-sensitive” preset of parameters. Reads that did not align to nuclear genome or aligned to mitocondrial genome were removed. PCR duplicates were removed using samtools. Then read positions were corrected for transposon insertion offset as described in (PMID:24097267).
Peak calling was performed using pooled samples by conditions and using MACS2 with the “--nomodel”, “--extsize 146”, “--qvalue 0.05”, "--nolambda" and "--keep-dup all" flags and arguments.
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: *.narrowPeak, list of the peaks called by MACS2
Submission date Jul 11, 2019
Last update date Mar 29, 2020
Contact name Chiara Balestrieri
Organization name IRCCS San Raffaele Scientific Institute
Department Center for Omics Sciences
Street address Via Olgettina 58
City Milan
ZIP/Postal code 20132
Country Italy
Platform ID GPL18573
Series (2)
GSE122946 A molecular network regulating the proinflammatory phenotype of human memory T lymphocytes
GSE134161 A molecular network regulating the pro-inflammatory phenotype of human memory T lymphocytes (ATAC-seq)
BioSample SAMN12256373
SRA SRX6432639

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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