GSM2865070: RPE ChIPSeq DMSO H3K4me1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H3K4me1
Cell type
Cell type Class
Unclassified
Cell type
Unclassified
NA
NA
Attributes by original data submitter
Sample
source_name
RPE
tisue
retina
disease
normal
chip antibody
H3K4me1 (Abcam, ab8895)
agent
DMSO
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
RPE cells were treated with largazole or vehicle for 16 hours and cross-linked with 1% formaldehyde for 15 minutes at room temperature (25°C). Cells were washed two times with PBS and membranes ruptured in hypotonic buffer (50 mM NaCl, 1% NP-40 alternative, 2 mM EDTA, 10 mM Tris, 1 mM DTT, 2 mM EDTA, 1X protease inhibitor cocktail (Roche # 04693124001). The cell nuclei were recovered by centrifugation and resuspended in lysate buffer (150 mM NaCl, 0.5% Triton X-100, 2 mM EDTA, 0.1% SDS, 20 mM Tris, 1 mM DTT, 1X protease inhibitor cocktail). Resuspended samples were sonicated for 25 cycles (30s 'on' at high level and 30s 'off' per cycle) using a Bioruptor (Diagenode; Denville, NJ, USA) and spun for 10 minutes at 16,000x g in a microcentrifuge. Samples were incubated for 5 hours at 4°C with 5 to 20 μg of antibodies and 20 μl of 50% slurry with protein A beads (Millipore; Billerica, MA, USA). The immunoprecipitated chromatin was then recovered and DNA purified using phenol chloroform extraction. ChIP sequencing libraries were prepared using an Illumina ChIP-Seq DNA Sample Prep Kit (cat. # IP-102-1001), with a starting sample varying from 2 to 20 ng of DNA isolated from the immunoprecipitation step. Libraries were prepared according to Illumina's protocol. Barcodes were assigned as follows: RPE_Input.fastq.gz;CCAACACT, RPE_ChIPSeq_DMSO_H3K27ac.fastq.gz;TCTTCGAC,RPE_ChIPSeq_largazole_37.5nM_H3K27ac.fastq.gz;CTTCCTTC,RPE_ChIPSeq_largazole_75nM_H3K27ac.fastq.gz;AACTTGCC,RPE_ChIPSeq_largazole_300nM_H3K27ac.fastq.gz;CGACCTAA, RPE_ChIPSeq_DMSO_H3K4me1.fastq.gz;GACTACGA,RPE_ChIPSeq_largazole_37.5nM_H3K4me1.fastq.gz;ACCATCCT, RPE_ChIPSeq_largazole_75nM_H3K4me1.fastq.gz;GTACACCT,RPE_ChIPSeq_largazole_300nM_H3K4me1.fastq.gz;TACATCGG, RPE_ChIPSeq_DMSO_RNAPII.fastq.gz;ACTCCTAC,RPE_ChIPSeq_largazole_75nM_RNAPII.fastq.gz;ACGAGAAC, RPE_ChIPSeq_largazole_300nM_RNAPII.fastq.gz;ATCGTCTC.