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| Status |
Public on Aug 30, 2019 |
| Title |
H3K36me2_ChIPseq_Cal27 |
| Sample type |
SRA |
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| Source name |
Cal 27 cells & S2 cells (spiked-in)
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| Organisms |
Drosophila melanogaster; Homo sapiens |
| Characteristics |
chip antibody: H3K36me2 (Cell Signaling Tech, #2901)
cell line: Cal27
cell type: Patient-derived head and neck squamous cell carcinoma cell line
genotype: NSD1 wild-type
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| Growth protocol |
Cal27 cells were cultured in Dulbecco's modified Eagles' medium (DMEM, Invitrogen) with 10% fetal bovine serum (FBS, Sigma).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with 1% formaldehyde (Sigma). Fixed cell preparations were washed, pelleted and stored at -80°C. Sonication of lysed nuclei (lysed in a buffer containing 1% SDS) was performed on a BioRuptor UCD-300 for 60 cycles, 10s on 20s off, centrifuged every 15 cycles, chilled by 4°C water cooler. Samples were checked for sonication efficiency using the criteria of 150-500bp by gel electrophoresis. After the sonication, the chromatin was diluted to reduce SDS level to 0.1% and concentrated using Nanosep 10k OMEGA (Pall). Before ChIP reaction 2% of sonicated drosophila S2 cell chromatin was spiked-in the samples for quantification of total levels of histone mark after the sequencing. ChIP reaction for histone modifications was performed on a Diagenode SX-8G IP-Star Compact using Diagenode automated Ideal ChIP-seq Kit. Dynalbeads Protein A (Invitrogen) were washed and then incubated with specific antibodies, and 1.5 million cells of sonicated cell lysate combined with protease inhibitors for 10 hr, followed by 20 min wash cycle with provided wash buffers. Reverse cross linking took place on a heat block at 65°C for 4 hr. ChIP samples were then treated with 2ul RNase Cocktail at 65°C for 30 min followed by 2ul Proteinase K at 65°C for 30 min. Samples were then purified with QIAGEN MiniElute PCR purification kit as per manufacturers’ protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse crosslinked and DNA was isolated following the same protocol.
KAPA HyperPrep Kit
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Raw ChIP sequencing reads were aligned using BWA version 0.7.17 with default parameters. After alignment, we sorted the bam files using samtools, then igvtools was used to generate wig files (igvtools count -w 25 -e 200). Finally, bigWig files were obtained using wigToBigWig.
Raw RNA sequencing reads were aligned using STAR version 2.5.3a with default parameters. The bigWig files were generated similarly as ChIP-seq.
Raw reads from whole-genome bisulfite sequencing were aligned using BWA (version 0.6.1). Low-quality sequence at the 3′ ends were trimmed. After alignment, we filtered duplicated or poorly mapping reads (>2% mismatches or aberrant insert size). Samtools (version 0.1.18) in mpileup mode was applied to call CpG methylation. We used igvtools to generate the tdf files.
Genome_build: hg19, mm10 and dm6 for human, mouse and drosophila data, respectively
Supplementary_files_format_and_content: bigWig, tdf
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| Submission date |
Aug 20, 2018 |
| Last update date |
Aug 30, 2019 |
| Contact name |
Carmen Rivas |
| E-mail(s) |
mcarmen.rivas@usc.es
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| Organization name |
Universidad de Santiago de Compostela
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| Department |
CIMUS
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| Lab |
P2L7
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| Street address |
Avda Barcelona
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| City |
Santiago de Compostela |
| ZIP/Postal code |
15706 |
| Country |
Spain |
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| Platform ID |
GPL25476 |
| Series (1) |
| GSE118785 |
H3K36me2 recruits DNMT3A and shapes intergenic DNA methylation landscapes |
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| Relations |
| BioSample |
SAMN09864094 |
| SRA |
SRX4579174 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3347557_Cal27_H3K36me2.bw |
491.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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