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| Status |
Public on Apr 16, 2016 |
| Title |
S_pombe_WT_PROseq |
| Sample type |
SRA |
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| Source name |
background: 972 h-
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
background: 972 h-
assay: PRO-seq
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| Growth protocol |
S. cerevisiae cultures were grown in YPD media from a starting OD600 of 0.2 to a final OD600 = 0.5. S. pombe cultures were grown in YES media with the same starting and ending OD600 as used for budding yeast.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Immediately prior to harvesting cultures, a fixed amount of spike-in culture (i.e. S.pombe into S. cerevisaie samples) was added to each sample culture for PRO-seq and RNA-seq experiments. For PRO-seq and PRO-cap assays, cultures were then spun down and washed once in ice-cold water. Samples were then spun again and resuspended in ice-cold permeabilization buffer (0.05% sarkosyl) and left on ice for 20 minutes. Permeabilized cells were then spun down at 400XG for 5 minutes. Permeabilized cell pellets were resuspeneded in 120 uL 2.5X transcription buffer (50 mM Tris-HCl, pH 7.7, 500 mM KCl, 12.5 mM MgCl2). To the reaction buffer we then added 3.75 uL of each biotin-11-NTP (epicentre), 6 uL .1M DTT, 3 uL SUPERase Inhibitor (invitrogen) and 141 uL DEPC treated water. The run-on was then performed by adding 15 uL 10% sarkosyl and placing samples at 30°C for 5 minutes. After the run-on, samples were spun and the reaction buffer was removed. RNA was then isolated using the hot acid phenol extraction protocol, followed by ethanol precipitation.
PRO-seq and PRO-cap Libraries were prepared according to Kwak, H., Fuda, N. J., Core, L. J., & Lis, J. T. Science (2013). mRNA-seq libraries were prepared using Illumina's TruSeq Stranded mRNA Sample Preparation LS protocol.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Nascent RNA
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| Data processing |
PRO-seq: Adaptor sequences were clipped from reads. Sequences were trimmed to a maximum length of 36 (minimum = 15). The reverse complement of reads was generated for alignment. Using Bowtie (version 1.0) Reads were first aligned to ribosomal DNA genomes of both S. cerevisiae and S. pombe. Any reads that failed to align to ribosomal DNA were then aligned to a combinded genome of S. cerevisiae and S. pombe. We allowed for a maximum of 2 mismatches Only uniquely aligning reads were used for downstream analysis. Unique reads were parsed based on their species of origin. Bedgraph files were created by recording only the most 3' base of each read (which represents the position of the Pol II active site). The counts at each position in a bedgraph file were normalized as counts per million mappable spike-in reads. Normalized bedgraphs were then converted to bigwig formated files.
Genome_build: S. pombe: ASM294v2; S. cerevisiae: sacCer3
Supplementary_files_format_and_content: processed files are in bigwig format. Each bigwig file contains normalized counts of either read 3'-ends (PRO-seq & mRNA seq), or read 5'-ends (PRO-cap).
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| Submission date |
Dec 18, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Thomas Booth |
| E-mail(s) |
[email protected]
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| Phone |
607-351-5499
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| Organization name |
Cornell University
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| Department |
Molecular Biology and Genetics
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| Lab |
John Lis
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| Street address |
526 Campus Road
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| City |
Ithaca |
| State/province |
New York |
| ZIP/Postal code |
14853 |
| Country |
USA |
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| Platform ID |
GPL13988 |
| Series (1) |
| GSE76142 |
Divergence of Transcription Regulation and the Function of a Conserved Elongation Factor in Budding and Fission Yeast |
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| Relations |
| BioSample |
SAMN04349998 |
| SRA |
SRX1490950 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1974985_3870_7157_12395_C53ARACXX_SP_WT_972h-COMBINED_REPS_GGCTAC_R1spikeNormed_minus.bw |
9.9 Mb |
(ftp)(http) |
BW |
| GSM1974985_3870_7157_12395_C53ARACXX_SP_WT_972h-COMBINED_REPS_GGCTAC_R1spikeNormed_plus.bw |
9.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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