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| Status |
Public on Aug 01, 2024 |
| Title |
HEK293, pSer2, T4A, rep1 |
| Sample type |
SRA |
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| Source name |
HEK293
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
cell type: Human embryonic kidney
chip antibody: pSer2 (Sigma, #MABE953)
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| Treatment protocol |
To prepare samples for pThr4 ChIP, we treated HEK293 cell lysate with Ssu72/symplekin complex at 4°C for 30 minutes before binding to the 6D7 antibody. To generate T4A and T4WT CTD samples for pSer2 immunoprecipitation, transient transfection of 12 μg of either plasmid was performed using PEI. Following transfection, 2.5 μg of alpha amanitin was added to cells for 48 hours. Finally, to prepare HA-RPRD1B samples, HEK293 were transfected with HA-RPRD1B using PEI (1:7 plasmid to reagent ratio) to overexpress the protein of interest.
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| Growth protocol |
DMEM, 10% fetal bovine serum, 1x penicillin/streptomycin. Cells were cultured at 37C at 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, for HA-tagged RPRD1B, HEK293 cells were double crosslinked with 2mM DSG for 15 min followed by secondary fixation with 1% formaldehyde for 10 min at room temperature. Single crosslinking was used for RPB1 ChIP using 1% formaldehyde for 10 min. Crosslinking was quenched with 0.125 M glycine for 5 min. Cells were successively lysed in lysis buffer LB1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1× PI), LB2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× PI) and LB3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, 1× PI). Chromatin was sonicated to an average size of ~200–500 bp using UCD-200 Biorupter (30s on and 30 s off for 30 min). A total of 5 μg of HA antibody (cat: C29F4), pThr4 antibody (Active Motif, cat: 61361), or pSer2 antibody (Sigma, SKU: MABE953) was pre-mixed in a 50μl volume of Dynabeads protein A or protein G (Invitrogen) and was added to each sonicated chromatin sample and incubated overnight at 4°C. The chromatin-bound beads were washed two times with low salt buffer (0.1% Na Deoxycholate, 1% Triton X-100, 1mM EDTA, 50mM HEPES pH 7.5, 150mM NaCl), once with high salt wash buffer (0.1% Na Deoxycholate, 1% Triton X-100, 1mM EDTA, 50mM HEPES pH 7.5, 500mM NaCl), once with LiCl wash buffer (250mM LiCl, 0.5% NP-40, 0.5% Na-Deoxycholate, 1mM EDTA, 10mM Tris-Cl pH 8.0) and twice in TE buffer. The chromatin was reverse crosslinked overnight at 65°C with shaking at 750rpm. After DNA extraction using phenol-chloroform, the DNA was resuspended in 10mM Tris-HCl pH 8.0.
The extracted DNA was used to construct the ChIP-seq library using the NEBNext Ultra II DNA Library Prep Kit followed by sequencing with an Illumina HiSeq 3000 or NovaSeq X Plus system.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq X Plus |
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| Data processing |
After initial assessment of read quality, pSer2 and RPRD1B (HA-tag) ChIP-Seq data was mapped onto human reference genome, hg38, with Bowtie2 v. 2.5.0 aligner for paired-end reads using default parameters. pThr4 single-end ChIP-Seq reads were mapped onto human reference genome, hg19, using BWA v.0.7.17.
Coverage tracks in .bigwig format were generated from filtered .bam files (mapq > 20).
TSS/TES profiling was done using plotProfile on matrices generated with 50-bp bins using the computeMatrix function from the deeptools v.2.2.3
After alignment, MACS2 v.2.2.7.1 in Galaxy (parameters: --broad; --broad-cutoff of q < 0.1 for pThr4 and HA-RPRD1B; pSer2 -default) was used to call peaks for IP-samples against Input.
Assembly: hg19 (pThr4), hg38 (pSer2, HA-RPRD1B)
Supplementary files format and content: bigWig, broadPeak
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| Submission date |
Mar 29, 2024 |
| Last update date |
Aug 01, 2024 |
| Contact name |
Svetlana Panina |
| E-mail(s) |
[email protected], [email protected]
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| Organization name |
The University of Texas at Austin
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| Department |
Molecular Biosciences
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| Street address |
101 East 21st St.
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| City |
Austin |
| State/province |
TX |
| ZIP/Postal code |
78712 |
| Country |
USA |
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| Platform ID |
GPL34284 |
| Series (1) |
| GSE262826 |
Thr4 phosphorylation on RNA Pol II occurs at early transcription regulating 3’-end processing |
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| Relations |
| BioSample |
SAMN40657963 |
| SRA |
SRX24105772 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8180079_pSer2_T4A_rep1.bigwig |
183.8 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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