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| Status |
Public on Jun 18, 2020 |
| Title |
ChIP-seq-Clone21-CTCF-CRISPR-CTCF-rep2 |
| Sample type |
SRA |
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| Source name |
HAP1
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| Organism |
Homo sapiens |
| Characteristics |
cell type: KBM7-derived near haploid cells
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| Treatment protocol |
HAP1 cells were co-transfected with the two components of the Sleeping Beauty transposon system: pSA-MCS-2kb, the transposon vector with the 2kb insert containing a CTCF binding site and the TSS of PARL gene, and the transposase vector, pCMV(CAT)T7-SB100. Clonal lines were established following transposon genome editing. Hi-C was then performed for two clones (Clone 21 and Clone 25) with the highest numbers of insertions. Where indicated, CRISPR editing using a gRNA targeting the CTCF binding site within ectopic insertions was performed, and Capture-C was used to characterize effects upon CTCF binding site disruption.
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| Growth protocol |
HAP1 cells (Carette et al., Nature 477, 340-343 (2011)), derived from KBM7 cells, a chronic myelogenous leukemia cell line, were cultured in IMDM with 10% FBS and 1% penicillin/streptomycin at 37°C with 5% CO2 (as described in Carette et al., 2011).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation (ChIP) was performed as previously described (Hsiung, et al., Genes Dev., 2016), using the H3K27Ac antibody (Active Motif 39685), CTCF antibody (Millipore 07-729), and Rad21 antibody (abcam ab992). Briefly, ~20 million HAP1 cells were fixed in 1% formaldehyde in fresh culture medium at room temperature for 10 minutes, followed by quenching in 1M glycine for 5 minutes. Crosslinked cells were lysed for 10 minutes in 1mL cold Cell Lysis Buffer (10mM Tris pH 8.0, 10mM NaCl, and 0.2% NP-40/Igepal), supplied with Protease Inhibitors (Sigma-Aldrich P8340) and phenylmethylsulfonyl fluoride (PMSF). Nuclei were pelleted, resuspended in 1mL room temperature Nuclei Lysis Buffer (50mM Tris pH 8, 10mM EDTA, 1% SDS), with Protease Inhibitors and PMSF, and were incubated on ice for 20 minutes. The samples were sonicated at 100% amplitude, 30 seconds on/30 seconds off, for 45 minutes, in a bath sonicator (QSonica Q800R3). Sonicated materials were centrifuged, with the supernatant subsequently collected, and diluted with 4mL IP Dilution Buffer (20mM Tris pH 8, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS), with Protease Inhibitors and PMSF. 50µL protein A/G agarose beads (Thermo Fisher 15918014, Thermo Fisher 15920010) and 50µg isotype-matched IgG control were added to sonicated chromatin to pre-clear it for >2 hours at 4°C. Beads were then spun down, with 200µL supernatant containing pre-cleared chromatin saved as “input” before immunoprecipitation. The remaining pre-cleared chromatin was split into equal volumes, each incubated with antibody or isotype-matched control (IgG) pre-bound protein A/G beads, and rotated overnight at 4°C. Chromatin-bound beads were washed on ice, once with IP Wash 1 (20mM Tris pH 8, 2mM EDTA, 50mM NaCl, 1% Triton X-100, 0.1% SDS), twice with High Salt Buffer (20mM Tris pH 8, 2mM EDTA, 500mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP Wash 2 (10mM Tris pH 8, 1mM EDTA, 0.25M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate), and twice with TE. Beads were then moved to room temperature, and were eluted twice with a total volume of freshly prepared 200µL Elution Buffer (100mM NaHCO3, 1% SDS). Into each IP and input, 12µL 5M NaCl and 2µL RNase A (10mg/mL, Roche through Sigma 10109169001) were added, and samples were incubated at 65°C overnight. 3µL proteinase K (20mg/mL, Roche through Sigma 3115879) was then added, for an additional 2 hours at 65°C. DNA was column cleaned up using a QIAquick PCR Purification Kit (QIAGEN 28106). For ChIP-sequencing, library construction was performed using Illumina’s TruSeq ChIP sample preparation kit (Illumina IP-202-1012), followed by size selection using SPRIselect beads (Beckman Coulter, B23318). Libraries were quality checked, quantified prior to 1 x 75 bp sequencing on the Illumina NextSeq 500.
See "extract protocol"
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ChIP-seq
ChIP-insertions_or_edited_endo_bedgraphs.tar
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| Data processing |
Raw sequencing reads were mapped to hg19 using Bowtie. SAM files were converted to BAM files using SAMtools. For transcription factor (or other targets with narrow peaks, such as CTCF and Rad21 here), MACS (Zhang et al., Genome Biol., 2008) was used for generating Wiggle and for peak calling. For histone marks (H3K27Ac), BAM files were converted to Wiggle using MACS and subsequently converted to bigWig. Subsequent peak calling was performed using Sicer (Xu et al., Methods Mol Biol., 2014). For the data tracks visualized in the figures, bamCoverage from Deeptools (Ramírez., Nucleic Acids Research, 2016) was used to generate counts-per-million (CPM)-normalized bigwig files from bam files. The CPM-normalized bigwig files were later converted to bedgraphs for insertion-proximal regions.
Basecalls using bcl2fastq2 v2.15.0.4, and parameters --barcode-mismatches 1
Mapping to reference genome hg19 canon with Bowtie 1.0.0 using parameters --chunkmbs 1024 -y -n 2 --best -k 1 --maxbts 800 -l 28 -e 80 --sam-nohead --sam
Wiggle generated with MACS using parameters --nomodel --shiftsize=120--format BAM --gsize 2615371906 --tsize 36 --bw 120 --mfold 12 --wig --space 1
For CTCF and Rad21 (factors with narrow binding profiles): peaks were called with MACS using MFOLD=12, PVALUE="" --format BAM --gsize $EFFGENSIZE --tsize 36 --bw 120 --mfold $MFOLD --wig --space 1 $PVALUE
Genome_build: Filter blacklist regions from peaks, and convert to broadpeak format (see UCSC Genome Browser for format specs)
Supplementary_files_format_and_content: bw alignment files
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| Submission date |
Jun 17, 2020 |
| Last update date |
Jun 19, 2020 |
| Contact name |
Ross Hardison |
| E-mail(s) |
rch8@psu.edu
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| Organization name |
Pennsylvania State University
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| Street address |
303 Wartik Lab
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE137376 |
Alteration of genome folding via engineered transposon insertion |
| GSE152721 |
Alteration of genome folding via engineered transposon insertion [CTCF RAD21 ChIP-seq] |
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| Relations |
| BioSample |
SAMN15307244 |
| SRA |
SRX8570933 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4625038_2608.hg19.broadpeak.gz |
995.6 Kb |
(ftp)(http) |
BROADPEAK |
| GSM4625038_2608.hg19.bw |
2.2 Gb |
(ftp)(http) |
BW |
| GSM4625038_2608.hg19.cpm.bw |
161.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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