|
| Status |
Public on Mar 30, 2017 |
| Title |
RNAseq_Spt5-T4.5-untagged_I |
| Sample type |
SRA |
| |
|
| Source name |
RNAseq_Spt5-T4.5-untagged
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: FWP484
growth_medium: EMM
treatment: thiamine + 1-Napthaleneacetic acid (NAA)
treatment time: 4.5 hr
spike_in: ERCC
demultiplex_code: GATCAG
spt5_gene_status: wild type
|
| Treatment protocol |
Spt5 depletion experiments were performed by adding NAA (0.5mM in DMSO, napthaleneacetic acid, Sigma, N0640) and thiamine (100nM, thiamine hydrochloride, Sigma, T4625), to cells grown to a density of 10^7 cells/ml (O.D.600 ~ 0.5). Cultures were incubated with shaking at 30°C for 4.5 hours.
|
| Growth protocol |
S. pombe strains were grown in EMM complete media at 30°C.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted using the hot phenol method (PMID: 12854975 )
For RNA-seq libraries, 200 µg of total RNA from each strain was spiked-in with 10 µl of 1:100 diluted ERCC control RNA(). Then, poly(A)+ RNA was enriched by two rounds of Dynabeads Oligo (dT)25 (Invitrogen) purification, followed by alkaline fragmentation and size selection of fragments 40-70 nucleotides. Size-selected RNA was dephosphorylated and ligated to an RNA linker at the 3’ end. First-strand cDNA was synthesized using a primer with sequence complementary to the 3’-linker and an additional adapter sequence, using Superscript III reverse transcriptase (Invitrogen). cDNA was circularized using CircLigase (Epicentre). The library was amplified with 8-14 cycles of PCR, using primers specific for the adaptor sequence and the products were gel purified. Library size distributions and concentrations were determined using an Agilent Bioanalyzer. All strains were done in duplicate. RNA-seq libraries were sequenced on the Illumina HiSeq platform at the Tufts University Core Facility.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
RNAseq_Spt5-T4-5-21104_I
Strain_FWP484_genotype_untagged
|
| Data processing |
Trimming with custom script of adapter CTGTAGGCACCATCAAT
Alignment with TopHat 2.0.10 with minimum and maximum intron lengths 20 and 5000, respectively (-i 20 -I 500”), to a 'genome' of ASM294v2 plus sequences of ERCC spike-ins.
Spike-in normalization factors were obtained by calculating all pairwise linear model slopes for ERCC spike-ins, then deconvoluting to a vector of normalization factors. Coverage values were linearly transformed by these scaling factors.
WIGs were produced by sampling every 10bp.
Genome_build: ASM294v2
Supplementary_files_format_and_content: WIG track files, sampled every 10bp on each strand.
|
| |
|
| Submission date |
Aug 03, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter J Park |
| E-mail(s) |
peter_park@harvard.edu
|
| Phone |
617-432-7373
|
| Organization name |
Harvard Medical School
|
| Department |
Center for Biomedical Informatics
|
| Street address |
10 Shattuck St
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL17225 |
| Series (2) |
| GSE85125 |
Genome-wide analyses reveal widespread roles for Spt5 in sense and antisense transcription [RNA-seq] |
| GSE85182 |
Genome-wide analyses reveal widespread roles for Spt5 in sense and antisense transcription |
|
| Relations |
| BioSample |
SAMN05507125 |
| SRA |
SRX1996823 |
