GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM1974988 Query DataSets for GSM1974988
Status Public on Apr 16, 2016
Title S_pombe_WT_PROcap
Sample type SRA
Source name background: 972 h-
Organism Schizosaccharomyces pombe
Characteristics background: 972 h-
assay: PRO-cap
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.
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.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
Description Nascent RNA
Data processing PRO-cap: Using Bowtie (version 1.0) Reads were first aligned to the ribosomal DNA genome. Any reads that failed to align to ribosomal DNA were then aligned to the genome of interest. We allowed for a maximum of 2 mismatches Only uniquely aligning reads were used for downstream analysis. Begraph files were created by recording only the position of the most 5' nucleotide for each read. Counts at each genome position were then normalized by the total number of mapple reads (RPM). Normalized bedgraphs were then converted to bigwig formatted 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).
Submission date Dec 18, 2015
Last update date May 15, 2019
Contact name Gregory Thomas Booth
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 GPL13988
Series (1)
GSE76142 Divergence of Transcription Regulation and the Function of a Conserved Elongation Factor in Budding and Fission Yeast
BioSample SAMN04350001
SRA SRX1490953

Supplementary file Size Download File type/resource 6.3 Mb (ftp)(http) BW 6.2 Mb (ftp)(http) BW 5.7 Mb (ftp)(http) BW 5.7 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap