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Sample GSM3576656

Query DataSets for GSM3576656

Status

Public on Jan 23, 2019

Title

100_K562_H3_R2

Sample type

SRA

Source name

K562 Cell line

Organism

Homo sapiens

Characteristics

tissue: K562 Cell line
tissue type: Cell Culture
ip target: H3
chip antibody: ab1791(Abcam)

Extracted molecule

genomic DNA

Extraction protocol

Conventional libraries: After precipitating and pelleting, DNA was dissolved in 30 μl of TE buffer pH 8.0. The recovered DNA was converted into libraries using NebNext Ultra II DNA library preparation kit, following manufacturer’s protocol.
TAF-ChIP: Fixed cells were directly sorted in 240 μl of 140mM RIPA (10mM Tris-Cl pH 8.0, 140mM NaCl, 0.1mM EDTA pH8.0, 1% Triton X-100, and 0.1% SDS), and sonicated with 3 cycles at low power settings for breaking the nuclei. 15 μl of Protein A and G Dynabeads were coupled to 1 μg of specific antibody in the blocking buffer (RIPA 140mM supplemented with 0.2 mg/ml BSA, 0.05 mg/ml of glycogen and 0.2 mg/ml of yeast t-RNA) for 3 hrs at 4°C. The unfragmented chromatin were centrifuged at 14000 rpm for 10 minutes at 4°C, and the supernatant was transferred to the tube with blocked and antibody coupled beads. The samples were incubated at 4°C overnight with head over tail rotations. The samples were then washed twice briefly with 300 μl of home made tagmentation buffer (20 mM Tris(hydroxymethyl)aminomethane pH 7.6; 10 mM MgCl2; 20% (vol/vol) dimethylformamide) using magnetic rack for beads separation. The washed beads were resupended in 20 μl of 1X tagmentation DNA buffer (Nextera XT Kit) containing 1 μl of Nextera DNA tagmentation enzyme and incubated at 37 °C for 40 minutes with constant shaking in a thermoblock at 500 rpm. Following the tagmentation, the beads were washed as following; once with 140mM RIPA (10mM Tris-Cl pH 8.0, 140mM NaCl, 0.1mM EDTA pH8.0, 1% Triton X-100, and 0.1% SDS), four times with 250mM RIPA (10mM Tris-Cl pH 8.0, 250mM NaCl, 0.1mM EDTA pH8.0, 1% Triton X-100, and 0.1% SDS) and twice with TE buffer pH8.0 (10mM Tris-Cl pH 8.0 and 0.1mM EDTA pH8.0). The samples after proteinase K treatment for reverse-crosslinking were subjected to phenol chloroform extraction. After precipitating and pelleting, DNA was dissolved in 30 μl of TE buffer pH 8.0. The DNA was amplified in a 100 μl reaction with 1X NEBNext High-Fidelity PCR Mix with primers containing molecular indices (listed in Table 1) with following program; 72 °C for 3min, {98 °C for 10 seconds, 63 °C for 30 seconds, 72 °C for 30 seconds} for 12 cycles, 72 °C for 5 minutes, and hold at 4 °C. The PCR reaction was purified with beads based size selection to remove any fragment that might be of larger than 1000 bp size. Ampure Xp beads were added to the PCR reaction in a ratio of 0.2X ratio to bind larger fragments. The magnetic beads were separated with the help of magnetic rack and supernatant was transferred to a fresh tube. Ampure Xp beads were added to the PCR reaction in a ration of 0.8X to bind the target library. After PCR purification, libraries were analyzed on Agilent Bioanalyzer for size distribution and the concentration was measured using Qubit fluorometer. The finished libraries were pooled in equimolar amounts and sequenced on illumina NextSeq 500.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

TAF ChIP from 100 K562 cells for H3 Replicate 2
Used as control while processing

Data processing

Library strategy: TAF-ChIP
De-multiplexing and fastq file conversion was performed using blc2fastq (v.1.8.4)
Mapping was performed using bowtie2 (v. 2.3.3) against ensembl release 84 of BDGP6 for Drosophila sample and K562 samples against hg38 (ensemble release 84).
The mapped bam files were normalized to RPKM using deepTools, and bigwig coverage file was generated
Genome_build: dm6 (Drosophila melanogaster), hg38 (K562 Cells)
Supplementary_files_format_and_content: UCSC compatible library size normalised bigwig tracks

Submission date

Jan 23, 2019

Last update date

Jan 26, 2019

Contact name

Junaid Akhtar

E-mail(s)

[email protected]

Organization name

University of Mainz

Department

Institute of Neurobiology and Developmental Biology