|
| Status |
Public on Apr 18, 2015 |
| Title |
xrn1∆, DMSO control, SAP treatment, Replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
xrn1-delta DMSO control, SAP treatment
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: MATalpha his3-delta1 leu2-delta0 lys2-delta0 ura3-delta0 xrn1::hygMX
treatment: DMSO, recombinant Shrimp Alkaline Phosphatase (Affymetrix)
|
| Treatment protocol |
At mid-log phase (OD600 ~0.6), cells were treated with Tunicamycin (Sigma) 2.5ug/uL or DMSO control
|
| Growth protocol |
Yeast cells were grown overnight in YEPD at 30ºC, then diluted back to OD600 0.15 and grown to mid-log phase before treatment
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was obtained via hot-phenol extraction, then treated with TURBO DNase (Ambion). oligo-dT beads (Life Technologies) were used to enrich for polyA RNA
RNAs with 5OH termini were ligated to a DNA/RNA hybrid oligo linker containing a 5' desthiobiotin, 8 nucleotide unique molecular index (UMI), and a 3'-phosphate using purified E. coli RtcB. Samples were electrophoresed on denaturing gel and excised. Samples were reverse transcribed with Protoscript II RT (NEB) and a degenerate primer, then purified with RNAClean XP Beads (Beckman-Coulter). DNA Polymerase I (Enzymatics), E Coli Ligase (Enzymatics), and RNAse H (NEB) were used to perform second strand synthesis. Following purification with AMPure XP beads (Beckman-Coulter), samples were purified with Streptavidin Dynabeads (Invitrogen). Samples were PCR amplified using barcoded Illumina Truseq primers and purified with SPRIselect beads (Beckman-Coulter). Samples were quantitated with Qubit before sequence submission.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Unique Molecular Index (UMI) trimmed and duplicates removed using Umitools
Alignment to SacCer1 using Bowtie
Calculation of 5' end coverage and conversion to bedGraph format using bedtools
Genome_build: SacCer1
Supplementary_files_format_and_content: Genome Coverage of 5' end of reads in bedGraph format
|
| |
|
| Submission date |
Sep 18, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Jay R. Hesselberth |
| E-mail(s) |
jay.hesselberth@cuanschutz.edu
|
| Organization name |
University of Colorado School of Medicine
|
| Department |
Biochemistry and Molecular Gentetics
|
| Lab |
Jay Hesselberth
|
| Street address |
12801 E 17TH AVE
|
| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
| |
|
| Platform ID |
GPL17143 |
| Series (1) |
| GSE61527 |
Capture and sequence analysis of RNAs with 5'-hydroxyl termini in S. cerevisiae |
|
| Relations |
| BioSample |
SAMN03073516 |
| SRA |
SRX703764 |
