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Sample GSM2140624

Query DataSets for GSM2140624

Status

Public on Dec 24, 2016

Title

WT1plusKTS cells replicate 3 input

Sample type

SRA

Source name

K562 cell line

Organism

Homo sapiens

Characteristics

cell line: K562
transfections apart from pef1abirav5-neo: pEF1a-Flagbiotin-(WT1+17AA/+KTS)-puro

Treatment protocol

Stable transfectants w pEF1aBirAv5-neo plasmid

Growth protocol

Cultured in RPMI 1640 medium w 10% FBS w 1 mg/ml G418

Extracted molecule

genomic DNA

Extraction protocol

After crosslinking with 1% formaldehyde for 10 minutes, chromatin shearing was done by sonication in a Bioruptor UCD-200 (Diagenode, Liège, Belgium). The resulting lysate was divided into one aliquot for streptavidin capture (the equivalent of 9-10 million cells), another for Histone 3 K4 tri-methylation (H3K4me3; ab 8580, Abcam, Cambridge, UK) immunoprecipitation (the equivalent of 1-2 million cells) and a third aliquot for non-immunoprecipitated lysate to use as input control. The H3K4me3 immunoprecipitation and the input control preparation were done using the MagnaChip kit, according to the manufacturer’s instructions, increasing the reaction volumes to accommodate the input cell number. For the streptavidin capture, Dynabeads M-280 Streptavidin (Invitrogen) were used, according to the manufacturer’s recommendations with minor modifications. Briefly, beads were washed with PBS three times prior to use, 3 mg of beads were used for each sample, incubation was done at room temperature, and washing after incubation was done five times with PBS with 0.1% BSA. After washing, the streptavidin beads were resuspended in Chip Elution Buffer from the MagnaChip kit and all samples proceeded to Proteinase K treatment and DNA purification according to the MagnaChip protocol.
ThruPlex DNA-seq kit (Rubicon Genomics, Ann Arbor, Michigan, USA) according to manufacturer’s recommendations.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

Illumina RTA 1.17.21.3 was used for base-calling
ChIP-seq reads were aligned to hg19 using Bowtie2-2.2.7 with parameters –local –no-unal
Peaks where called using MACS2-2.1.1.20160226 with default settings
Genome_build: hg19
Supplementary_files_format_and_content: BED files with the found peaks

Submission date

May 02, 2016

Last update date

May 15, 2019

Contact name

Linnea Järvstråt

E-mail(s)

[email protected]

Organization name

Lund University

Department

Department of Hematology and Transfusion Medicine