GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1541714

Query DataSets for GSM1541714

Status

Public on Jun 04, 2015

Title

by_sd_2_p

Sample type

SRA

Source name

grown in SD; same paired-end library in 3 lanes

Organism

Saccharomyces cerevisiae

Characteristics

strain: BY4741
genotype/variation: Mat a; his3delta1leu2delta0 ura3delta0 met15delta0
protocol: 5PSeq
spike-in: S. Pombe + IVTs
media: SD complete

Treatment protocol

For cycloheximide (CHX) treatment, CHX was added to 0.1mg/mL final and incubated 5 min at 30ºC. For S. pombe cells CHX was incubated either 5 or 30 min. When combining CHX treatment with H2O2 stress (final 0.2mM H2O2), samples were transferred to watery ice at the specified stress time points and incubated for extra 5 min at 4ºC in presence of 0.1mg/mL CHX final. For 3-amino-1,2,4-triazole (3-AT) treatment S96 strain (MATa lys5∆ gal2∆, S288c background) was grown in CSM-His to mid-log phase (OD600~1). Cells were diluted to OD600~0.15 in either CSM-His (control) or CSM-His + 100mM 3-AT (treated) and harvested at OD600~0.5. For polyribosome fractionation yeast cells were incubated for 5 min with CHX at 0.1mg/mL final and then broken with glass beads using a FastPrep machine in presence of Lysis buffer (20 mM Tris-HCl pH 8, 140 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 100µg/mL CHX, 500µg/mL heparin and supplemented with EDTA-free Complete protease inhibitor). Fractions were separated by ultracentrifugation (2h 40min at 88244 g) using a 10-50% sucrose gradient, the presence of RNA was estimated by measuring Abs at 254 nm.

Growth protocol

Strains were grown to mid-log phase (OD~1) at 30ºC. S. cerevisiae was gown in YPD (1% yeast extract, 2% peptone, 2% glucose), Complete Synthetic Media (SD) or Synthetic Media without Histidine (SD-His) S. pombe (h-) was grown using YES media (0.5% yeast extract, 3% glucose, supplemented with 225 mg/l of adenine, histidine, leucine, uracil and lysine hydrochloride).

Extracted molecule

polyA RNA

Extraction protocol

All samples were collected by centrifugation and frozen in liquid N2. Total RNA was isolated by a standard hot phenol method and treated with RNase-free DNaseI using Turbo DNA-free kit (Ambion)
We used 10-100 µg of DNA-free total RNA as starting material. As an internal control, we added polyadenylated in vitro transcripts (ATCC 87482, 87483 and 87484, that were modified to be enriched respectively in 5?cap, 5?PPP or 5?P ends) and S. pombe RNA. For 5?P sequencing (5PSeq), samples were directly subjected to RNA ligation. To generate a negative control of 5?P molecules (5PSeq-fragmented) RNA sample was chemically fragmented by incubating the samples at 80ºC for 5 min in the presence of RNA fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM KOAc, 30 mM MgOAc). Fragmented 5?OH sites were then phosphorilated by treatment with 5 unit of T4 Polynucleotide Kinase (T4 PNK, NEB) for 1 hour at 37ºC in the presence of RNase inhibitor (RNasin +, Promega). The sample was phenol:chloroform purified and ethanol precipitated and subjected to RNA ligation. From the ligation step we used the same protocol all the methods. The treated RNA sample was incubated overnight at 16ºC with 20 units of T4 RNA ligase 1 (NEB) in the presence of 10 mM DNA/RNA rP5_RND oligo (CTTTCCCTACACGACGCTCTTCCGATrCrUrNrNrNrNrNrNrNrN). RNA integrity was controlled by RNA Bioanalyzer and then polyA purified using Oligotex (Quiagen) according to manufacturer?s instructions. For the ribosomal depleted samples (5µg total RNA), polyA purification was substituted by treatment with Ribo-Zero Magnetic Gold Kit (Epicentre). PolyA enriched RNA was fragmented at 80ºC for 5 min in the presence of RNA fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM KOAc, 30 mM MgOAc). RNA was retrotranscripted with Superscript II (Life Technologies) and primed with random hexamers. The retrotranscription reaction was incubated for 10 minutes at 25ºC, 5 minutes at 42ºC and heat inactivated for 15 minutes at 72ºC. Second cDNA strand was perform by a unique cycle of PCR (1 min at 98ºC; 2 min at 50ºC and 15 at 72ºC) using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) and priming with BioNotI-P5-PET (Biotin-TATAGCGGCCGCAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT). From this point libraries were generated as previously described (PMID:23295673). Briefly double stranded cDNA was first purified using HighPrep beads (Magbio) and then bound to Dynabeads M-280 Streptavidin beads (Life Technologies) according to manufacturers instructions. Bound DNA molecules were successively washed and subjected to Ends repair, A addition and adaptor ligation using Nebnext DNA Library Prep Master Mix (NEB). 0.5µL of common adapter (2.5µM) was ligated to each sample. The common adapter was prepared by annealing P7MPX_linker_for and P7MPX_linker_rev. Beads were washed and subjected to PCR amplification (30s 96ºC; 15-18 cycles (30s 96ºC; 30s 65ºC; 30s 72ºC); 5min 72ºC) using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) and 0.1µM final of PE1.0 and the appropriate indexed PE2_MTX PCR oligo. Sample was size selected using 0.6x-0.9x (v/v) HighPrep beads (Magbio) to 300-500 bp. Libraries were sequenced (single-end) using either an Illumina HiSeq 2000 or a MiSeq. The first experiments were indexed adding a barcoded P7 linker (as in PMID:23295673) and subjected to a paired-end sequencing where first read contains the 8nt molecular barcode followed by the 5?sequence and the reverse read contains a 6 nt index followed by and A and the reverse complementary sequence of the identified transcripts.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

CAGE

Instrument model

Illumina MiSeq

Description

processed data file: by_sd_p.txt, pomb_p.txt

Data processing

Base-calling was done during processing of sequencing data with the illumina pipeline casava v1.8.2
Random barcode was extracted for each read
Reads were mapped to a merged genome (S. cerevisiae genome (SGD R64-1-1), S. pombe ASM294v2.20 and IVT sequences) using the Novoalign software with default settings
Reads with multiple alignments, or mapping quality <30, or carrying soft-clipped bases on the 5’ end were excluded from downstream analysis
5' end of reads passing quality filtering were extracted; total number of reads per genome location were calculated; total unique number of reads (or unique molecule, based on random barcode sequences) per genome location were calculated
Genome_build: Saccharomyces cerevisiae S288C reference genome R64-1-1 (sacCer3), downloaded from SGD(http://www.yeastgenome.org/). Schizosaccharomyces pombe reference genome ASM294v2.20, downloaded from pombase.org (http://www.pombase.org/).
Supplementary_files_format_and_content: Processed data contain the genomic location of the identified unique identified molecules after controlling for presence of molecular barcodes (unum) and reads per million (rpm) for each biology replicate.

Submission date

Nov 08, 2014

Last update date

May 15, 2019

Contact name

Wu Wei

E-mail(s)

[email protected]

Organization name

EMBL Heidelberg

Department

Genome Biology Unit