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| Status |
Public on Aug 17, 2015 |
| Title |
ChIP-seq of 10 million K562 cells with GATA1 antibody - replicate 2 |
| Sample type |
SRA |
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| Source name |
K562 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
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| Growth protocol |
K562 cells were cultured in RPMI medium supplemented with 10% FCS and antibiotics. They were analyzed with a CASY cell counter to determine cell numbers. Peripheral blood was obtained from healthy volunteers as approved by the ethics committee at the Medical University of Vienna. Coagulation was prevented with EDTA or heparin, peripheral blood was diluted 1:1-1:3 in PBS, and peripheral blood mononuclear cells (PBMCs) were isolated with Lymphoprep density gradient (Axis-Shield) following manufacturer instructions. Purified cells were suspended in RPMI supplemented with 10% FBS and penicillin-streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Three alternative chromatin immunoprecipitation (ChIP) protocols were successfully tested in combination with ChIPmentation. These protocols use different fixation, sonication, lysis, and washing conditions, making it possible to use ChIPmentation with essentially any ChIP-grade antibody; ChIP version 1 (H3K4me3, H3K27me3): Cells were washed once with PBS and fixed with 1 % paraformaldehyde in up to 1 ml PBS for 5 minutes at room temperature. Glycine was added to stop the reaction. Cells were collected at 500 x g for 10 minutes at 4 °C and washed twice with up to 1 ml ice-cold PBS supplemented with 1 μM PMSF. The pellet was lysed in Cell Lysis Buffer (50mM HEPES/KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 % Glycerol, 0.5 % NP-40, 0.25 % Triton X-100, 1x protease inhibitors (Sigma)) for 10 minutes on ice. Nuclei were isolated by spinning the lysed cells for 10 minutes at 1000 x g at 4 °C, the supernatant was discarded and the pellet was resuspended in Sonication Buffer (10 mM Tris-HCl pH 7.6, 1mM EDTA, 0.1 % SDS) and sonicated in a 130 μl microTUBE (for up to 3 x 106 cells) on a Covaris S220 for 12 minutes until most of the fragments were 200-700 base pairs long (settings: duty cycle 2 %, peak incident power 105 Watts, cycles per burst 200). Lysates were centrifuged at full speed for 5 minutes at 4 °C and the supernatant was transferred to a new tube. The lysate was diluted to 200 μl per IP to a buffer composition of 20 mM HEPES, 0.1 % SDS, 1 %Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and incubated with an antibody against H3K4me3 (1 μg/IP, Diagenode C15410003-50) or H3K27me3 (1 μg/IP, Millipore 07-449) over night at 4 °C on a rotator. 20 μl of Protein A Magnetic Beads were blocked over night with 0.1 % BSA in PBS and added to the IP the next day for 2 hours on a rotator at 4 °C to capture the immunoprecipitated fragments. The immunoprecipitated chromatin was washed subsequently with WBI (20mM HEPES, 150mM NaCl, 0.1 % SDS, 0.1 % DOC, 1 % Triton X-100, 1 mM EDTA, 0.5mM EGTA) (twice), WBII (20 mM HEPES, 500 mM NaCl, 0.1 % SDS, 0.1 % DOC, 1 % Triton X-100, 1 mM EDTA, 0.5 mM EGTA) (once), WBIII (20 mM HEPES, 250 mM LiCl, 0.5 % DOC, 0.5 % NP-40, 1 mM EDTA, 0.5 mM EGTA) (once) and WBIV (20 mM HEPES, 1 mM EDTA, 0.5 mM EGTA) (twice). Then beads were incubated with 70 μl elution buffer (0.5 % SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl pH 8.0) containing 2 μl of Proteinase K (NEB). Beads in Elution Buffer were incubated for 1 hour at 55 °C and 8 hours at 65 °C to revert formaldehyde crosslinking, and supernatant was transferred to a new tube. Another 30 μl of elution buffer was added to the beads for 1 minute and eluates were combined and incubated with another 1 μl of Proteinase K for 1 hour at 55 °C. Finally, DNA was purified with SPRI AMPure XP beads (ratio sample:beads 1:2) or Qiagen MinElute columns; ChIP version 2 (H3K4me1, and H3K36me3 and REST): Cells were washed once with PBS and fixed with 1 % paraformaldehyde in up to 1.5 ml PBS for 10 minutes at room temperature. Glycine was added to stop the reaction. Cells were collected at 500 x g for 10 minutes at 4 °C and washed twice with up to 1 ml μl ice-cold PBS supplemented with 1 μM PMSF. The pellet was lysed in RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 140 mM NaCl, 1 % Triton x-100, 0.1 % SDS, 0.1 % DOC, 1x protease inhibitors (Sigma)) and sonicated in a 1 ml milliTUBE in a Covaris S220 for 30 minutes until most of the fragments were 200-700 base pairs long (settings: duty cycle 5 %, peak incident power 140 Watts, cycles per burst 200). Lysates were centrifuged at full speed for 5 minutes at 4 °C. In the meantime, 50 µl beads (10 µl for low-input ChIPmentation) were blocked and conjugated to an antibody by washing and resupsending them 2 times in PBS/0.5 % BSA/0.5 % Tween-20). A specific antibody was added and bound to the beads by rotating > 1 h at room temperature. Antibodies used in this study were H3K4me1 (1 µg/IP, Diagenode pAb-194-050), H3K36me3 (1 µg/IP, Diagenode pAb-192-050) and REST (10 µg/IP, Millipore 07-579). The supernatant was transferred to a 0.5 PCR tube and per ChIP 50 μl of blocked antibody conjugated magnetic protein A beads were added and incubated for 3 hours at 4 °C. Immunoprecipitation beads were washed subsequently with 150 μl RIPA (twice), RIPA-500 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 500 mM NaCl, 1 % Triton x-100, 0.1 % SDS, 0.1 % DOC,) (twice), RIPA-LiCl (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 250 mM LiCl, 1 % Triton x-100, 0.5 % DOC, 0.5 % NP40) and TE pH 8.0 (twice). Then beads were then incubated with 70 μl elution buffer (0.5 % SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl pH 8.0) containing 2 μl of Proteinase K (NEB). Beads in Elution Buffer were incubated for 1 hour at 55 °C and 8 hours at 65 °C to revert formaldehyde crosslinking, and supernatant was transferred to a new tube. Finally, DNA was purified with SPRI AMPure XP beads (ratio sample:beads 1:2) or Qiagen MinElute columns; ChIP version 3 (H3K27ac, PU.1, CTCF and GATA1): Cells were washed once with PBS and fixed with 1% paraformaldehyde in up to 1.5 ml PBS for 5-10 minutes at room temperature. Glycine was added to stop the reaction. Cells were collected at 500 x g for 10 minutes at 4 °C and washed twice with up to 1 ml μl ice-cold PBS supplemented with 1 μM PMSF. The pellet was lysed in buffer L3B (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 x protease inhibitors (Sigma)) and sonicated in a 1ml milliTUBE in a Covaris S220 for 20 minutes until most of the fragments were 200-700 base pairs long (settings: duty cycle 5 %, peak incident power 140 Watts, cycles per burst 200). Lysates were supplemented with 1 % Triton-X-100 and centrifuged at full speed for 5 minutes at 4 °C. In the meantime, beads were blocked and conjugated to an antibody by washing them 2 times in PBS/0.5 % BSA and resuspending 50 μl of beads per IP (10 µl beads for low-input ChIPmentation) in 200 μl of PBS/0.5 % BSA. A specific antibody was added and bound to the beads by rotating >1h at room temperature. Antibodies used in this study were H3K27ac (2 µg, Diagenode pAb-196-050) PU.1 (5 μg/IP, Santa Cruz sc-352), CTCF (10 μl/IP, Millipore 07-729) and GATA1 (4 µg/IP and 2µg for low-input, Abcam ab11852). The supernatant was transferred to a 0.5 PCR tube and per ChIP 50 μl of blocked antibody conjugated magnetic protein A beads were added and incubated for 3 hours at 4 °C. Immunoprecipitation beads were washed subsequently with 150 μl TF-WBI (20 mM Tris-HCl/pH 7.4, 150 mM NaCl, 0.1 % SDS, 1 % Triton X-100, 2 mM EDTA) (twice), TF-WBIII (250 mM LiCl, 1 % Triton X-100, 0.7 % DOC, 10 mM Tris-HCl, 1 mM EDTA) (twice) and TET (0.2 % Tween-20, 10 mM Tris-HCl/pH 8.0, 1 mM EDTA) (twice). Then beads were incubated with 70 μl elution buffer (0.5 % SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0) containing 2 μl of Proteinase K (NEB). Beads in Elution Buffer were incubated for 1 hour at 55 °C and 8 hours at 65 °C to revert formaldehyde crosslinking, and supernatant was transferred to a new tube. Another 30 μl of elution buffer was added to the beads for 1 minute and eluates were combined and incubated with another 1 μl of Proteinase K for 1 hour at 55 °C. Finally, DNA was purified with SPRI AMPure XP beads (ratio sample:beads 1:2) or Qiagen MinElute columns.
Standard ChIP-seq library preparation: Purified ChIP DNA was end-repaired using the NEBNext End Repair Module (NEB) according to manufacturer’s instruction. Clean-up was done using Ampure XP beads (Agencourt) according to manufacturer’s instruction. Fragments were A-tailed using Klenow (3′→ 5′ exo-) polymerase (Enzymatics), and TruSeq-compatible adapters were ligated using T4 DNA Ligase (Enzymatics). The final library was size-selected using Ampure XP beads to remove adapter dimers.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
ChIP-seq of 10M K562 cells with GATA1 antibody - replicate 2
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| Data processing |
Illumina Casava1.7 software was used for basecalling.
Sequenced reads were trimmed for adaptor and Nextera sequences
Reads were mapped to hg19 whole genome using bowtie2 v2.2.4 with the –very-sensitive parameter
Duplicate reads were marked and removed with picard tools version 1.118
For ATAC-seq, ChIPmentation and ChIP-Tagmentation samples, reads aligning to the plus strand were offset by +4 bp, and reads aligning to the minus strand were offset by -5 bp
Read were extended to the average fragment size and bigWig files containing counts of reads per basepair per million created
Genome_build: hg19
Supplementary_files_format_and_content: Fastq files contain raw reads, bigWig files contain counts of reads per basepair per million
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| Submission date |
Jul 02, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Christoph Bock |
| E-mail(s) |
cbock@cemm.oeaw.ac.at
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| Organization name |
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
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| Street address |
Lazarettgasse 14
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| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE70482 |
ChIPmentation: fast, cheap, low-input ChIP-seq for histones and transcription factors |
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| Relations |
| BioSample |
SAMN03838378 |
| SRA |
SRX1080003 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1782703_K562_10M_ChIP-seq_GATA1_nan_nan_2_1_hg19.bigWig |
182.5 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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