|
| Status |
Public on Jan 19, 2011 |
| Title |
WT_mRNA-seq |
| Sample type |
SRA |
| |
|
| Source name |
yeast BY4741, mid-exponential phase
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741
genotype/variation: RPB3-3xFLAG::NAT his3D1::TEF2-GFP-Adh1 KAN
library strategy: NET-Seq
molecule: mRNA
ip: none
|
| Growth protocol |
Yeast strains were grown in YEPD at 30°C with shaking from an initial OD of 0.1 to mid-log phase with an OD of 0.6-0.8.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA purification from clarified lysate was done by the hot acid phenol method. Polyadenylated mRNA was purified from 50 µg total RNA using magnetic oligo-dT DynaBeads (Invitrogen).
Purified RNA was fragmented by alkaline hydrolysis and size selected by electrophoresis on a polyacrylamide gel to yield lightly fragmented RNA of size distribution similar to that of nascent RNA. Fragmented mRNA was dephosphorylated by T4 polynucleotide kinase (NEB). An preadenylated RNA linker was ligated onto the 3’ end of the immunoprecipitated RNA. Fragmentation of the ligated samples by alkaline hydrolysis followed by a size selection after electrophoresis on a polyacrylamide gel allowed for the final DNA library to contain inserts of a narrow range. cDNA synthesis by reverse transcription (SuperScriptsIII (Invitrogen)) was performed with a long 5’ phosohorylated primer split by two 18 carbon spacers. Circularization of the RT product was performed with CircLigase (Epicentre) according to the manufacturer’s directions. The PCR was performed directly on the circularized product resulting in DNA with Illumina cluster generation sequences on each end and a sequencing primer binding site positioned so that sequencing would start at the 3’ end. DNA was purified from a PCR reaction that had not reached saturation (~10 cycles) by band extraction from a polyacrylamide gel that had not reached saturation. DNA was sequenced on the Illumina Genome Analyzer 2 according to the manufacturer’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Total RNA purification from clarified lysate was done by the hot acid phenol method. Polyadenylated mRNA was purified from 50 µg total RNA using magnetic oligo-dT DynaBeads (Invitrogen).
|
| Data processing |
Image data obtained by the Illumina Genome Analyzer 2 was analyzed using the GAPipeline to extract raw sequences. Matrix and phasing parameters were estimated from a φX control lane. Raw sequences 40 bases long were composed of the cDNA of the fragmented RNA sequence. For RNA fragments smaller than 40 bases, the sequence is followed by part of the 5’ Illumina linker sequence which was removed in silico. Alignments to the yeast genome was performed by the alignment program, Bowtie 0.12.0 (http://bowtie-bio.sourceforge.net/). Bowtie settings were chosen so that three mismatches were allowed and alignments were required to be unique. The shortest sequenced fragments were approximately 18 nucleotides due to the RNA size selection step after ligation and random fragmentation. Alignments were first performed against tRNA and rRNA sequences to remove them. The remaining sequences were aligned against a recent version of the yeast genome downloaded from the Saccharomyces Genome Database (SGD, http://www.yeastgenome.org/) on October 11, 2009
|
| |
|
| Submission date |
Nov 03, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
L. Stirling Stirling Churchman |
| Organization name |
University of California-San Francisco
|
| Street address |
1700 Fourth St. Byers Hall - Room 404
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL9377 |
| Series (1) |
| GSE25107 |
Native elongating transcript sequencing (NET-seq) of wild type Saccharomyces cerevisiae and of DST1, RCO1, SET1, SET2, EAF3 deletion strains |
|
| Relations |
| SRA |
SRX031060 |
| BioSample |
SAMN00120240 |
