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Sample GSM4552118

Query DataSets for GSM4552118

Status

Public on May 15, 2020

Title

Hela_NT_FANCM_ChIP

Sample type

SRA

Source name

FANCM ChIP-Seq

Organism

Homo sapiens

Characteristics

cell line: Hela/GFP-DONSON cells
chip antibody: Bethyl A302-637A, Rabbit anti FANCM

Treatment protocol

Non-treated cells

Growth protocol

GFP-DONSON expressing HeLa cells 30 were cultured with L-Glutamine (Gibco), 200 ug/ml Hygromycin B (Invitrogen), and 5 ug/ml Blasticidin (Gibco). To induce GFP-DONSON expression, cells were incubated with 1 μg/ml Doxycycline for 48 hr.

Extracted molecule

genomic DNA

Extraction protocol

Cells were crosslinked with 1% formaldehyde in culture media for 8 min, followed by quenching the formaldehyde with 0.1 M glycine. Cells were washed with PBS, harvested by scraping, then suspended in lysis buffer (0.5% SDS, 10 mM EDTA, 50 mM Tris-HCL pH 8.0) supplemented with protease and phosphatase inhibitors. Lysates were sonicated in a 4 ⁰C water bath ultrasonicator (Bioruptor, Diagenode). The time of sonication was adjusted to yield short DNA fragments <500 bp (total 8 minutes, 30 seconds sonication, then cool 30 seconds). In some experiments the time was adjusted to yield longer DNA fragments of 500-5000 bp (2 x 30 seconds with a 30 second cooling period). Diluted lysates were incubated overnight at 4 °C with antibodies as indicated. Immunoprecipitations were performed using Protein G magnetic beads (Pierce, 10% v/v), or GFP Trap (Chromotek, gta-20). Bead bound complexes were washed with low salt immune complex buffer (0.1% SDS, 1% Triton x-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), high salt immune complex buffer (0.1% SDS, 1% Triton x-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), LiCl immune complex buffer (0.25 M LiCl, 1% NP-40, 1% mM EDTA, 10 mM Tris-HCl pH 8.0) and TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0). DNA was eluted in elution buffer (1% SDS, 0.2 M NaCl) with Proteinase K (100 µg/ml) overnight at 65 °C. Eluted DNA was purified with DNA Clean & Concentrator PCR purification Kit (ZYMO Research, D4033) according to the manufacturer instructions.
Illumina sequencing adapters with a T-overhang were ligated to the precipitated ChIP DNA fragments or the input DNA, with a corresponding A-overhang, to construct a sequencing library according to the manufacturer’s protocol (Illumina, San Diego, CA). The fragments were purified using a magnetic bead protocol and eighteen cycles of PCR amplification were performed to enrich for fragments with an adapter on both ends. The products were purified again with size selection (approximately 200–600 bases) using a dual bead selection protocol with SPRIselect Beads (Beckman Coulter, Brea, CA). These libraries were sequenced on an Illumina Hi-Seq 2500 sequencer using on-board cluster generation on a rapid run paired end flow cell for 75 X 75 cycles (DONSON) and single end of 75 bp for 75 cycles (FANCM). Real-time analysis was performed using RTA v1.18.66.3 and base-calling was performed using bcl2fastq v2.18.0.12.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Description

NT_IP3_FANCM_Input_bwCompare_log2.bw

Data processing

ChIP-seq reads were aligned to the human genome assembly hg38 using BWA [version 0.7.17] using the following parameters
Picard [version 2.20.8] SamFormatConverter was used to convert SAM to BAM, SortSAM was used to sort the resulting BAM file [VALIDATION_STRINGENCY=Lenient], MarkDuplicates was used to mark duplicates
Deeptools [version 3.3.0] bamCoverage was used to generate BigWig files with the following parameters [-bs 150 -e --normalizeUsing RPKM --ignoreDuplicates]
Deeptools [version 3.3.0] bigwigCompare was used to calculate log2 ratios between bigwig files from each ChIP-seq and the corresponding Input [parameters: --skipZeroOverZero --operation log2 --skipNonCoveredRegions --outFileFormat bigwig --blackListFileName]
Genome_build: hg38
Supplementary_files_format_and_content: bigwig, bedgraph

Submission date

May 14, 2020

Last update date

May 15, 2020

Contact name

Marina A Bellani

E-mail(s)

marinab@nih.gov

Organization name

NIA

Department

LMB