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| Status |
Public on Jun 15, 2022 |
| Title |
504_pA-RNA_2 |
| Sample type |
SRA |
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| Source name |
rrp6d
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain number: 504
genotype: rrp6d
target molecule: p(A) RNA
ip antibody: none
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| Growth protocol |
Cells were grown in rich YES medium.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
50 ml yeast cultures with an OD600 of 1.2 were cross-linked with 1% formaldehyde (Roth) for 15 min at room temperature. The reaction was quenched with 125 mM glycine for 5 min. The frozen pellet was resuspended in 500 µl lysis buffer (250 mM KCl, 1x Triton-X, 0.1% SDS, 0.1% Na-Desoxycholate, 50 mM HEPES pH 7.5, 2 mM EDTA, 2 mM EGTA, 5 mM MgCl2, 0.1% Nonidet P-40, 20% Glycerol) with 1 mM PMSF and Complete EDTA free Protease Inhibitor Cocktail (Roche). Lysis was performed with 0.25-0.5 mm glass beads (Roth) and the BioSpec FastPrep-24 bead beater (MP-Biomedicals), 8 cycles at 6.5 m/s for 30s and 3 min on ice. DNA was sheared by sonication (Bioruptor, Diagenode) 35 times for 30 s with a 30 s break. Cell debris was removed by centrifugation at 13 000 x g for 15 min. The crude lysate was normalized based on the RNA and Protein concentration (Nanodrop, Thermo Scientific) and incubated with 1.2 µg immobilized (Dynabeads Protein A, Thermo Scientific) antibody against Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody - ChIP Grade (ab5095, abcam) for at least 2 h at 4°C. The resin with immunoprecipitates was washed five times with each 1 ml of lysis buffer and eluted with 150 µl of elution buffer (50 mM Tris HCl pH 8.0, 10 mM EDTA, 1% SDS) at 65°C for 15 min. Cross-linking was reversed at 95°C for 15 min and subsequent RNase A (Thermo Scientific) digest for 30 min followed by Proteinase K (Roche) digest for at least 2 h at 37°C. DNA was recovered by phenol-chloroform-isoamylalcohol (25:24:1, Roth) extraction with subsequent ethanol precipitation.
For sequencing, a ChIP-seq library was made using the NEBNext Ultra II DNA Library Prep Kit for Illumina kit (NEB).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Sequencing reads obtained in the poly(A) RNA sequencing (pA RNA), Pol II ChIP, nascent RNA sequencing (Pol II RIP) and total RNA sequencing experiments were mapped to the S. pombe reference genome (PomBase, release 2018) using splice-aware alignment tool STAR version 2.7.3a (Dobin et al., 2013). Alignment of RIP-seq and RNA-seq data was performed with STAR default parameters. Unspliced alignments of ChIP-seq data was enforced through parameters ‘--alignIntronMax 1’ and ‘--alignEndsType EndToEnd’. Reads mapping to ribosomal RNA have been removed from further analysis.
Genomic read counts were obtained using a custom script that extended basic htseq-count functionality with ‘--mode intersection-strict’ option. Additionally, for RNA assays pA RNA, Pol II RIP and total RNA, we used the ‘--stranded yes’ option to disambiguate which strand reads originated from. In short, reads mapping uniquely to protein-coding genes and reads that map less than 16 times were counted and normalized by gene length and sequencing depth (TPM, Transcripts Per Million). We chose 16 (dg/dh have in 12-13 copies) as multi-mapping threshold for heterochromatic genes to eliminate low-complexity reads without discarding reads originating from heterochromatic regions. In the read counting, we did not distinguish between individual gene copies but used one representative copy to collect all reads stemming from any one copy.
Assembly: PomBase, release 2018
Supplementary files format and content: tab-delimited text files include raw counts for each sample
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| Submission date |
Mar 25, 2022 |
| Last update date |
Jun 15, 2022 |
| Contact name |
Mario Halic |
| E-mail(s) |
[email protected]
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| Organization name |
St. Jude Children’s Research Hospital
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| Department |
Structural Biology
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| Street address |
262 Danny Thomas Place
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| City |
Memphis |
| State/province |
Tennessee |
| ZIP/Postal code |
38105 |
| Country |
USA |
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| Platform ID |
GPL22682 |
| Series (1) |
| GSE186273 |
Ccr4-Not complex reduces transcription efficiency in heterochromatin |
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| Relations |
| BioSample |
SAMN26973946 |
| SRA |
SRX14617385 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5972818_504_pA-RNA_2_pombe_gene_count_matrix.csv.gz |
94.1 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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