|
| Status |
Public on Jan 04, 2018 |
| Title |
Lsk1as_CDK9as_DMSOr2 |
| Sample type |
SRA |
| |
|
| Source name |
Lsk1as_CDK9as_DMSO
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: cdk9as lsk1as: cdk9T120G::hphMX6 lsk1F353G::kanMX6 leu1-32 ura4-D18 his3-D1 ade6-M210 h+
treatment: DMSO
treatment duration: 5 minutes
|
| Treatment protocol |
10 mL cultures were placed into 15 mL falcon tubes, treated with 10 μM 3-MB-PP1 or an equivalent volume of 100% DMSO. Treated cultures were left shaking at 30 °C for specified amount of time. Treatments were terminated by pouring cultures into 30 mL ice cold water and then immediately pelleting cells.
|
| Growth protocol |
S. pombe cultures were grown in YES media from a starting OD600 of 0.2 to a final OD600 = 0.5 before treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Immediately prior to treatments, a fixed amount of spike-in culture (i.e. S. cerevisaie samples) was added to each sample culture for PRO-seq experiment. After treatment, 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 libraries were prepared according to Mahat et al. Nature Protocols (2016).
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Nascent RNA
ePROseq_Pombe_Lsk1asCDK9as_DMSO_COMBINED_pombe_SpikeNormed_plus.bw
ePROseq_Pombe_Lsk1asCDK9as_DMSO_COMBINED_pombe_SpikeNormed_minus.bw
|
| Data processing |
Library strategy: PRO-seq
Adaptor sequences were clipped from read 3' ends. In cases where inline barcodes were used, they were clipped from 5' ends. 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: S288C_reference_genome_R64-1-1_20110203
Supplementary_files_format_and_content: processed files are in bigwig format. There are two bigwig files for each sample, one for each strand. Each bigwig file contains normalized counts of read 3'-ends.
|
| |
|
| Submission date |
Aug 07, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Gregory Thomas Booth |
| E-mail(s) |
gregtbooth@gmail.com
|
| Phone |
607-351-5499
|
| Organization name |
Cornell University
|
| Department |
Molecular Biology and Genetics
|
| Lab |
John Lis
|
| Street address |
526 Campus Road
|
| City |
Ithaca |
| State/province |
New York |
| ZIP/Postal code |
14853 |
| Country |
USA |
| |
|
| Platform ID |
GPL20584 |
| Series (1) |
| GSE102308 |
Cdk9 regulates a promoter-proximal checkpoint to modulate RNA Polymerase II elongation rate in fission yeast |
|
| Relations |
| BioSample |
SAMN07455722 |
| SRA |
SRX3066731 |
