GSM2753123: mm atac immatureb.1; Mus musculus; ATAC-seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
ATAC-Seq
Antigen
ATAC-Seq
Cell type
Cell type Class
Blood
Cell type
B cells
NA
NA
Attributes by original data submitter
Sample
source_name
ImmatureB
cell type
ImmatureB
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
To prepare Nuclei, respective flow sorted mouse or EBV transformed human peripheral B cells (1x105) cultured in RPMI media (with L- glutamine, 10% FBS, 1mM Sodium pyruvate, 10mM HEPES, 100units/ml penicillin, 100µg/ml Streptomycin) were centrifuged at 500g for 5 min, which was followed by a wash with ice-cold PBS and centrifugation at 500g for 5 min. Cells were lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500g for 10 min at 40C and supernatant removed. The nuclei pellet was resuspended in the transposase reaction mix (25 μL 2X Tagment buffer, 2.5 μL Tagment DNA enzyme (Illumina, FC-121-1030) and 22.5 μL nuclease-free water). The transposition reaction was carried out at 37 °C for 30 min. Directly following transposition the sample was purified using a Qiagen MinElute kit. Following purification, we amplified library fragments using Nextera PCR Primers (Illumina Nextera Index kit) and NEBnext PCR master mix (New England lab, 0541) for a total of 10-12 cycles. The libraries were then purified using a Qiagen PCR cleanup kit. The amplified, adapter ligated libraries were size selected using Life Technologies' E-Gel® SizeSelect™ gel system in the 150-650bp range. The size-selected libraries were quantified using the Agilent Bioanalyzer and via q-PCR in triplicate using the KAPA Library Quantification Kit on the Life Technologies Step One System. Libraries were sequenced on the Illumina Hiseq2500 system to generate 75-100M, 50bp paired end reads.