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| Status |
Public on May 08, 2024 |
| Title |
K562, wtLSD1GFP_antiH3K14ac-rep2,CUT&RUN |
| Sample type |
SRA |
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| Source name |
K562
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| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
cell type: human immportalized chronic myelogenous leukemia
cut&run antibody: Millipore Sigma, #07-353
treatment: parental wtLSD1-GFP
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| Treatment protocol |
Parental K562 cells are homozygously edited with LSD1-GFP. Edited K562 cells are heterozygously edited with Y391K-LSD1-GFP (2 alleles with Y391K-LSD1-GFP and 1 allele with wt-LSD1-GFP)
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| Growth protocol |
RPMI1640, 10% fetal bovine serum, 2 mM L-glutamine, 1x penicillin/streptomycin. Cells were cultured at 37 degrees C and 8% CO2
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Protocol was adapted from Epicypher's CUTANA CUT&RUN Protocol (https://www.epicypher.com/resources/protocols/cutana-cut-and-run-protocol/). Briefly, K562 cells were harvested through centrifugation, bound to ConA beads, permeabilized with digitonin, incubated with antibodies, washed, incubated with pAG-Mnase in the presence of CaCl2, and quenched with EDTA/EGTA along with 0.5 ng of E. coli spike-in DNA to extract DNA.
CUT&RUN libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, #E7645S) complemented with NEBNext Multiplex Oligos for Illumina Index Primers Set 1 to 4 (New England Biolabs). 0.5 ng to 11 ng of DNA was used as starting material for all the samples. Libraries were amplified using 14 cycles on the thermalcycler. Post amplification libraries were selected at 250-700 bp in length using AMPure XP beads (Beckman Coulter, #A63880). Libraries were validated using Tapestation (Agilent D1000).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
CUT&RUN data processing was mostly adopted from the established protocol (Zheng Y et al (2020). Protocol.io, CUT&Tag Data Processing and Analysis Tutorial). FastQ data files were initially processed by fastp (v. 0.23.4), and quality checks were assessed by FastQC (v. 0.11.9). The paired-end reads were aligned to the human reference genome (GRCh38 assembly) using Bowtie2 (v. 2.4.4) as well as the E. coli genome (K-12 MG1655).
For GRch38 assembly, the following arguments were used: bowtie2 -p 8 --end-to-end --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700 -x /path-to-genome/ /GRCh38_noalt_as. For E. coli, the following arguments were used: bowtie2 -p 8 --end-to-end --very-sensitive --no-overlap --no-dovetail --no-mixed --no-discordant --phred33 -I 10 -X 700 -x /path/Escherichia_coli_K_12_MG1655/NCBI/2001-10-15/Sequence/Bowtie2Index/genome.
The sam output files were converted to the bam format using Samtools (v. 1.17).
For peakcalling, bam files were first converted to bed and bedgraph files using Bedtools (v. 2.31.0) and SEACR relaxed mode85 was used to call peaks by normalizing against the IgG control bedgraph files for each replicate. For CUT&RUN signal visualization, bam files were sorted by the coordinates and indexed using Samtools (v. 1.17), and the sorted bam files were converted to the bigwig files using bamCoverage application from the Deeptools package (v. 3.5.2).
RPGC normalization mode using the effective Genome size of 3049315783 bp and bin size of 1 was applied (bamCoverage -b "$file" -p 10 --binSize 1 --normalizeUsing RPGC --effectiveGenomeSize 3049315783 --extendReads --outFileFormat bigwig). The bigwig files were visualized by using Integrative Genomics Viewer (IGV) and analyzed using computeMatrix and plotProfile applications from the Deeptools package (v. 3.5.2).
Assembly: hg38
Supplementary files format and content: bigWig, peak files (except for IgG control)
Library strategy: CUT&RUN
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| Submission date |
Sep 14, 2023 |
| Last update date |
May 08, 2024 |
| Contact name |
Kwangwoon Lee |
| E-mail(s) |
klee76@bwh.harvard.edu
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| Organization name |
Brigham and Women's Hospital
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| Department |
Medicine
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| Lab |
P. A. Cole
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| Street address |
77 Avenue Louis Pasteur, NRB 164
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL30173 |
| Series (1) |
| GSE243231 |
Uncoupling Histone Modification Crosstalk by Engineering Lysine Demethylase LSD1 |
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| Relations |
| BioSample |
SAMN37397055 |
| SRA |
SRX21780086 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7781191_10_wtLSD1GFP_antiH3K14ac-rep2.bw |
108.6 Mb |
(ftp)(http) |
BW |
| GSM7781191_10_wtLSD1GFP_antiH3K14ac_rep2_SEACRrelaxed.bed.gz |
4.8 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
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