GSM5278717: GR Dex15min ChIPseq [GR 15MIN.1]; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
NR3C1
Cell type
Cell type Class
Unclassified
Cell type
Unclassified
NA
NA
Attributes by original data submitter
Sample
source_name
GR_Dex15min_ChIPseq
cell line/type
A1-2 breast cancer cells
treatment
15min Dex
antibody
Novus MBP2-42221
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For ChIPseq, cells were fixed in PBS with 1% methanol-free formaldehyde (Thermoscientific 28906) at 37C for 10 min and quenched with glycine. Cell pellets were resuspended in MNase swelling buffer (25 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, 5 mM Sodium Butyrate, 0.5 mM PMSF, and protease inhibitor cocktail (PIC) (ThermoFisher Scientific, 78429)) for enzymatic fragmentation or into hypotonic buffer (10 mM HEPES-NaOH pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 340 mM sucrose, 10% glycerol, 0.1% Triton X-100, 5 mM Sodium Butyrate, 0.5 mM PMSF, and PIC) for mechanical sonication. Cells were incubated on ice for 10 min then subjected to Dounce homogenization using 20-strokes (Duran Wheaton Kimble, 357542). For enzymatic fragmentation with MNase, nuclei were sedimented through digestion buffer (15mM Hepes pH 7.9, 60mM KCl, 15mM NaCl, 0.32M Sucrose, 5 mM Sodium Butyrate, 0.5 mM PMSF, and PIC) by centrifugation for 7 min at 930g (4C). Nuclei pellets were fully resuspended in digestion buffer containing 3.3 mM CaCl2 and digested with MNase (0.7U/1x10^6 cells, Worthington, cat#) at 37C for 15min. MNase fragmentation was stopped by addition of 10 mM EGTA and incubation for 5 min on ice. Digested nuclei were lysed in LB3 buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N- Lauroylsarcosine, 1% Triton X-100, 5 mM Sodium Butyrate, 0.5 mM PMSF, and PIC) using 5-passages through a 25g ( 0.508 mm x 1.6 cm) needle and brief sonication using 4 cycles (30 sec on/off, power high, 4C) with a Bioruptor sonicator (source). MNase digested chromatin lysate was recovered after 15 min centrifugation at 13,000g (4C). For mechanical sonication, nuclei pellets were resuspended in Shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF, 5 mM Sodium Butyrate, 0.1% SDS, and PIC) and chromatin was fragmented by sonication using the Covaris S220 (Peak Power, 175; Duty Factor, 10; Cycles/Burst, 200; Total Time, 600 sec). Sonicated chromatin was recovered by centrifugation at 20,000g (4C) for 10 min. 4C-seq was performed according to the published protocol (Krijger et al., 2020). Briefly, batches of 5-10 million cells were fixed in 2% Formaldehyde solution and flash frozen in liquid nitrogen. A single pellet of 5-10 million cells was used for each 4C replicate. Chromatin was first digested with NlaIII, ligated with T4 DNA Ligase to generate the 3C template, and purified with NucleoMag PCR beads. The purified 3C template was subsequently digested with DpnII, ligated with T4 DNA Ligase to generate the 4C template, and again purified with NucleoMag PCR beads. ChIP-seq libraries were generated using the ACCEL-NGS® 2S PLUS DNA library kit according to manufacturer's protocol and sequenced on the Illumina NextSeq platform. 4C-seq libraries were generated by two-step PCR using Expand Long Template PCR kit. The first step PCR reaction used reading and non-reading primers to amplify 4C fragments and was purified with a 0.8X AMPure XP purification. The second step PCR reaction added sequencing adapters and indexes and was purified using Qiagen PCR purification kits. Libraries were sequenced on an Illumina MiSeq with a target of 1-2 million single-end, 100bp reads per library.