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Sample GSM5057387

Query DataSets for GSM5057387

Status

Public on Jul 04, 2022

Title

RL1902_input_DoxMut

Sample type

SRA

Source name

MCF-7 cell line - ZF-D3A-mut

Organism

Homo sapiens

Characteristics

cell line: MCF-7
genotype: ZF-D3A-mut

Growth protocol

complete media with 1000 ng/ml doxycyline for 3 days

Extracted molecule

genomic DNA

Extraction protocol

Cells were washed two times with 10 ml of PBS and crosslinked for 5 minutes in 50 mM HEPES-KOH, pH 7.5, 100 mM NaCl, 1mM EDTA, 1% formaldehyde. The crosslinking reaction was quenched by the addition of glycine to a final concentration of 125 mM and washed twice with phosphate buffered saline (PBS). All subsequent solutions were supplemented with a protease inhibitor cocktail (Sigma Cat. # P8340). Cells were scraped off the plates with a rubber policeman in 10 ml of PBS and centrifuged at 3,000 rpm for 5 minutes in a swinging bucket rotor. The cell pellets were resuspended in 10 ml of 50 mM HEPES-KOH, pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, incubated on ice for 10 minutes and centrifuged at 3,000 rpm for 10 minutes in a swinging bucket rotor. Cell pellets were washed twice by gently adding 10 mM Tris-HCl, pH 8.1, 200 mM NaCl, 1 mM EDTA to the cell pellets, trying not to disturb the pellets, and centrifuged at 3,000 rpm for 5 min. Finally the cell pellets were resuspended in 0.1% SDS, 1 mM EDTA and transferred to a Covaris TC12x12 tube. The chromatin was sheared using a Covaris S2 sonicator with the following settings: time 12 min, duty cycle 5%, intensity 4, cycles per burst 200, temperature 4˚C, power mode frequency sweeping. Triton X-100 and NaCl were added to a final concentration of 1% and 150 mM respectively. The sheared chromatin was centrifuged at maximum speed in a microfuge for 15 minutes at 4˚C and the supernatant was transferred to a new tube.
Genomic DNA was fragmented with a Covaris S2 sonicator to a mean length of 200 bp, then end-repaired, A-tailed, ligated to methylated Illumina TruSeq adapters, bisulfite converted and subjected to 7 cycles of PCR amplification with KAPA HiFi Uracil+ DNA polymerase (KAPA Biosystems).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NovaSeq 6000

Description

input_DoxMut

Data processing

CASAVA v1.8.2
The WGBS sequenced reads were first trimmed using bbduk, and overlapping pairs were merged using BBMerge(Bushnell, Rood, and Singer 2017). The reads were mapped to the hg19 genome including the lambda and pUC19 sequences using BS-Seeker2 with Bowtie 2 as back-end aligner (-e 300 -X 2000)(Guo et al. 2013). The duplicated reads were removed using Sambamba markdup for paired-end reads and PALEOMIX for merged reads(Schubert et al. 2014). We then used CGmapTools to generate methylation calls(Guo et al. 2018).
Sequenced ATAC-seq reads were then trimmed with bbduk for Nextera adapters and mapped to the genome using Bowtie 2. Duplicated reads were removed and blacklisted regions filtered as with ChIP-seq, and peaks were called using MACS2 with “--nomodel -f BAM --keep-dup all” parameters. Replicates were merged using IDR, and each sample was validated visualising the insert size distribution following nucleosome periodicity
ChIP-seq reads were trimmed with bbduk(Bushnell, n.d.) and mapped to the genome using Bowtie 2(Langmead and Salzberg 2012) allowing for an insert site of 2000 bp, reads were deduplicated using Sambamba markdup function(Tarasov et al. 2015) and reads mapping to blacklisted genomic regions were filtered out using SAMtools(H. Li et al. 2009). We then used MACS2 to call peaks with the matched chromatin input sample as background for each ZF-D3A HA ChIP-seq experiment, specifying the parameters “-f BAMPE -q 0.05 --down-sample”(Feng et al. 2012). The peaks for replicate pairs were merged using IDR (https://github.com/nboley/idr)(Q. Li et al. 2011).
The RNA-seq reads were trimmed using fastp with default parameters(Chen et al. 2018), then mapped with HISAT2 (--rna-strandness RF) to an index including hg19 genome assembly and ERCC spike-in sequences(Sirén, Välimäki, and Mäkinen 2014). Then, RNA-seq read-pair counts were read into R using summarizeOverlaps function within the GenomicAlignments package and mapped to the UCSC hg19 gtf(Lawrence et al. 2013).
Genome_build: hg19 UCSC
Supplementary_files_format_and_content: Bisulfite-seq files (CGmap files) are the direct output from BSseeker2/CGmaptools (https://github.com/BSSeeker/BSseeker2), they contain chromosome position, (1) chromosome (2) nucleotide on Watson (+) strand (3) position (4) context (CG/CHG/CHH) (5) dinucleotide-context (CA/CC/CG/CT) (6) methylation-level = #_of_C / (#_of_C + #_of_T). (7) #_of_C (methylated C, the count of reads showing C here) (8) = #_of_C + #_of_T (all Cytosines, the sum of reads showing C or T in that position).
Supplementary_files_format_and_content: NarrowPeak files are the output of MACS2 software, ZF_Counts_table_GEO.tsv.gz was computed mapping RNA-seq with HISAT2 and extracting the counts with "featureOverlaps" in R.

Submission date

Feb 01, 2021

Last update date

Jul 04, 2022

Contact name

Ryan Lister

E-mail(s)

ryanlister@gmail.com

Phone

61864884407

Organization name

The University of Western Australia