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| Status |
Public on May 05, 2023 |
| Title |
HeLa CENP-A TAP, siCHAF1B, Rb CENP-A |
| Sample type |
SRA |
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| Source name |
HeLa
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
cell type: epithelial cells
genotype: TAP tagged CENP-A OE
treatment: hCHAF1B siRNA (Dharmacon J-019937-08-0005)
cut&run antibody: CENP-A (rabbit, Millipore #07-574)
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| Treatment protocol |
siRNA transfections were performed using Lipofectamine RNAiMax reagent according to manufacturer's instructions and cells were collected 72h post siRNA transfection
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| Growth protocol |
cells were grown in DMEM supplemented with 10% fetal bovine serum and 1% pen/strep at 37°C incubator with 5% CO2
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each CUT&RUN sequencing experiment, 4 x 10^5 siRNA treated HeLaCENP-A-TAP cells were harvested at RT and resuspended in Wash Buffer (20mM HEPES-NaOH pH 7.5, 150mM NaCl, 0.5mM spermidine, 1X Roche protease inhibitor cocktail). HeLaCENP-A-TAP cells were incubated with 10uL Concanavalin A-coated magnetic beads (BioMag) in Binding Buffer (20mM HEPES-KOH pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2 for 7m with rotation. The bead-cell slurry was permeabilized in Antibody buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.0025% digitonin, 2 mM EDTA, 0.1% BSA, 100 nM TSA, 0.1 unit/ml citrate synthase, 1 mM Oxaloacetic acid, 1x Roche protease inhibitor cocktail) and incubated with anti-CENP-A antibody (Antibody 1 = Millipore 07-574, Antibody 2 = Invitrogen MA1-20832) or IgG as a control in Antibody Buffer overnight at 4℃. Following two washes in Digitonin Buffer, beads were incubated in 50uL Digitonin Buffer (20mM HEPES-NaOH pH 7.5, 150mM NaCl, 05mM spermidine, 0.0025% digitonin, 1X protease inhibitor cocktail ) with 1x CUTANA pAG-MNase (EpiCypher 15-1016) for 1h at 4℃. The beads were washed twice in Digitonin Buffer followed by a wash in Low-Salt Wash Buffer (20mM HEPES-NaOH pH 7.5, 0.5mM spermidine, 0.0025% digitonin, 1X protease inhibitor cocktail). The beads were then incubated with ice-cold Calcium Incubation Buffer (3.5 mM HEPES-NaOH pH 7.5, 10 mM CaCl2, 0.0025% digitonin) for 30 min at 0℃. Beads were resuspended with EGTA-Stop Buffer (170 mM NaCl, 20 mM EGTA, 0.0025% digitonin, 50 μg/ml RNase A, 50pg/ml CUTANA E. coli Spike-in DNA – EpiCypher 18-1401) and incubated for 30 min at 37℃. To extract the DNA, 200ul of Oligo Binding Buffer (Zymo) was added to each sample and the total volume was then added to a Zymo-Spin DCC-5 column and centrifuged. The column was washed twice with DNA Wash buffer from the DNA Clean & Concentrator-5 Kit (Zymo). The DNA was eluted from the column in 15ul of DNA Elution Buffer and sample concentration was measured using a Qubit 3.0 fluorometer.
CUT&RUN libraries were prepared using an NEBNext Ultra II Library Kit for Illumina and NEBNext Multiplex Oligos for Illumina (Dual Index Primer Set 1) with modifications to manufacturer's protocol. Modifications: End Prep heat inactivation step was conducted at 50°C for 60 min. TheNEBNext Adaptor was diluted 1:25 in 10 mM Tris–HCl, pH 8.0 with 10 mM NaCl. After Adaptor Ligation, a DNA cleanup using 1.75× volumes of Sera-Mag Select beads was conducted. The PCR cycle conditions were programmed as following: 1 cycle: 98°C for 30s, 12cycles: 98°C for 10s, 65°C for 30s, 1 cycle: 65°C for 5m, Hold:4°C. After PCR amplification, DNA cleanup was conducted twice with 1.2× volumes of Sera-Mag Select (Cytiva) beads. Quality control was conducted on the resulting 15uL elution using Agilent 4200 TapeStation D1000 ScreenTape to determine sample concentration and sample quality.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Data processing |
Raw BCL output files were processed and demultiplexed using Illumina BaseSpace application Generate FASTQ version 1.0.0. Reads were trimmed from 150bp to 50bp using Illumina BaseSpace application FASTQ Toolkit v2.2.0.
Reads were trimmed for adaptor sequences using trimmomatic v0.33 with option –phred33 and then aligned to hg38 (https://www.ncbi.nlm.nih.gov/data-hub/genome/GCF_000001405.40/), CHM13v2 (https://www.ncbi.nlm.nih.gov/data-hub/genome/GCF_009914755.1/) and E.coli K12 (https://www.ncbi.nlm.nih.gov/data-hub/genome/GCF_000005845.2/) genomes, using bowtie2 v1.1.2 with options –v 2.
PCR duplicates were removed with picard MarkDuplicates v1.131.
CUT&RUN profiles were then calibrated by spike-in normalization using E. coli DNA as the internal reference or spike-in, analogous to that done for ChIP-seq (Meers et al. Elife. (2019). PMID: 31232687) to determine the normalization coefficient (NC). Spike-in normalized BigWig files were generated with deepTools bamCoverage -scaleFactor (NC) with defaults values.
Assembly: T2T-CHM13v2 and hg38
Supplementary files format and content: bigWig
Library strategy: CUT&RUN
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| Submission date |
Mar 14, 2023 |
| Last update date |
May 05, 2023 |
| Contact name |
Daniel Foltz |
| Organization name |
Northwestern University
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| Street address |
303 E Superior St
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
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| Platform ID |
GPL20795 |
| Series (1) |
| GSE227373 |
Histone H3/H4 chaperone CHAF1B prevents the mislocalization of CENP-A for chromosomal stability |
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| Relations |
| BioSample |
SAMN33761280 |
| SRA |
SRX19678802 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7099142_PV22_Ceto_T2t.normBG.bw |
160.6 Mb |
(ftp)(http) |
BW |
| GSM7099142_PV22_Ceto_hg38.normBG.bw |
124.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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