GSM3484566: BCL6ff Cre- BAT 30 deg C ChIP Input Control; Mus musculus; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Adipocyte
Cell type
Brown adipocytes
NA
NA
Attributes by original data submitter
Sample
source_name
interscapular brown adipose tissue
strain
BCL6-floxed (C57BL/6J)
tissue
interscapular brown adipose tissue
Sex
male
antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Interscapular brown adipose tissue was isolated, finely minced with a razor blade, and fixed in 1% formaldehyde in PBS for 20 minutes at room temperature, followed by addition of 125 mM glycine. Fragments were collected in cell lysis buffer (50 mM Tris (pH 8.0), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitor cocktail) and homogenized in a Dounce homogenizer (15 strokes with type B pestle). Following centrifugation, the nuclear pellet was resuspended in nuclear lysis buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 0.2% SDS, protease inhibitor cocktail) and incubated for 20 minutes on ice. Samples were sonicated with an XL-2000 sonicator (QSonica) for 12 rounds of 20 seconds on setting 2, with 60 seconds on ice between rounds, to generate fragments of 150-300 bp. Sonicated samples were centrifuged, and the supernatants were diluted two-fold with Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris (pH 8.1), 167 mM NaCl, protease inhibitor cocktail) and incubated with 2 micrograms of anti-H3K27ac antibody (Abcam #ab4729) overnight at 4 deg C, then with 50 microliters of Protein G Dynabeads (Invitrogen) for 4 hours at 4 deg C. Beads were washed on a magnetic stand twice with Buffer 1 (0.1% SDS, 0.1% sodium deoxycholate, 1% Triton x-100, 0.15 M NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0), once with Buffer 2 (same as Buffer 1 but with 0.5 M NaCl), once with Buffer 3 (0.25M LiCl, 0.5% sodium deoxycholate, 0.5% NP-40, 1 mM EDTA, 20 mM Tris-HCl, pH 8.0), twice with Buffer 4 (1 mM EDTA, 20 mM Tris-HCl, pH 8.0), and resuspended in elution buffer (50 mm Tris (pH 8.0), 10 mm EDTA, 1% SDS). Beads in elution buffer were incubated at 65 deg C for 10 minutes, and the supernatant collected. Elution was repeated a second time and the combined eluates were incubated at 65 deg C overnight to reverse crosslinks. Samples were treated with RNAse A (1 hour at 37 deg C), Proteinase K (2 hours at 65 deg C), and DNA was isolated with a Qiaquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions. DNA concentrations were measured with a Qubit dsDNA High Sensitivity Assay Kit and a Qubit 2.0 fluorimeter (Invitrogen). For construction of ChIP-Seq libraries, 10 ng of ChIP DNA was treated with end repair enzymes (Fisher Scientific) before proceeding to the 3' adenylation and adapter ligation steps of the TruSeq Stranded mRNA Library Prep Kit (Illumina) protocol. Subsequently, samples were resolved on a 2% agarose gel containing SYBR Gold Stain, visualized by ultraviolet illumination, and gel sections containing 250-500 bp fragments were excised and DNA extracted with a Gel DNA Recovery Kit (Zymoclean). Libraries were then amplified by PCR (15 cycles) and a library size of 250-500 bp was verified by agarose gel electrophoresis. Library concentration was measured with a Qubit dsDNA High Sensitivity Assay Kit and a Qubit 2.0 fluorimeter (Invitrogen). Libraries were pooled, denatured with NaOH, diluted to 1.8 pM, and sequenced on a NextSeq 500 Sequencing System (Illumina) with a NextSeq 500/550 High Output v2 kit (75 cycles) in single-read mode.