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Sample GSM1467866

Query DataSets for GSM1467866

Status

Public on Aug 09, 2014

Title

trf4 deletion

Sample type

SRA

Source name

Cbc2-associated RNA from meiotic cells

Organism

Saccharomyces cerevisiae

Characteristics

strain background: SK1
genotype/variation: trf4 deletion
Stage: meiosis
time: 6 hours
molecule subtype: Cbc2-associated RNA

Growth protocol

For synchronised sporulation, cells from 15% glycerol stocks were plated onto YPGly (YP+2% glycerol) plates and grown over night. Cells were transferred to YP4D (YP+4% glucose) plates and grown for 4-6 hours before inoculating 25ml YPD cultures. Cultures were grown over night at 30deg, and diluted the following afternoon into YPA (YP+2% KOAc) at 2.6x10^6 cells/ml or 5x106 cells/ml for wt and trf4Δ cells respectively. Cells were grown for ~18 hours at 30deg with shaking at 200 rpm, then washed and re-suspended in SPO medium (3g/L KOAc, 5mg/L uracil, 5mg/L histidine, 25mg/L leucine, 12.5mg/L tryptophan, 200mg/L raffinose) at 4x10^7 cells/ml and incubated at 25deg with shaking at 250rpm.

Extracted molecule

total RNA

Extraction protocol

1.6x10^10 sporulating cells were harvested and washed with PBS. The cells were resuspended in one pellet volume of lysis buffer (50mM HEPES pH7.5, 50mM KCl, 5mM MgCl2, 1mM DTT, cOmplete Mini EDTA-free Protease Inhibitors) and frozen drop-wise on liquid nitrogen. Cells were ground to a fine powder in a pestle and mortar under liquid nitrogen and thawed in a 50 ml tube on ice. The lysate was centrifuged for 5 min at 4500 rpm and the supernatant clarified by centrifugation for 20 min at 30,000g. Cleared lysate was transferred to a 15 ml tube and incubated with 100µl of IgG sepharose beads (G.E. Healthcare) for 2 hours at 4degC with gentle agitation. Beads were washed five times for 10 minutes at 4degC with IP Buffer (10mM Tris pH7.5, 120mM NaCl, 5mM MgCl2, 0.1% NP-40, 1mM DTT). Beads were incubated for 2 hours at 16degC with 100µl IP buffer and 1µl AcTEV protease (Invitrogen). Beads were removed by centrifuging through a Micro Bio-Spin column (Bio-rad), and the flow-through was diluted with 500µl IP buffer + 2mM CaCl2 and incubated for 50min at 4degC with 100µl calmodulin sepharose beads (G.E. Healthcare). Beads were washed three times for five minutes with IP buffer + 2mM CaCl2 and protein was eluted in 100µl calmodulin elution buffer (10mM Tris pH7.5, 120mM NaCl, 5mM EGTA). Beads were removed by centrifuging through a Micro Bio-Spin Column, and RNA was extracted by phenol:chloroform extraction and ethanol precipitation.
RNA was fragmented in 200mM Tris acetate pH 8.2, 500mM KOAc, 150mM MgOAc2 for 3 min at 94deg, ethanol precipitated and reverse transcribed from random hexamers using Superscript II (Life Technologies) according to manufacturer’s instructions. Tris pH7.8 and MgCl2 were added to 50mM and 5mM respectively, and second strand synthesis was performed with 2U RNase H (NEB) and 50U DNA polymerase I (NEB) at 16deg for 2.5 hours. Ends were repaired using 15U T4 DNA polymerase (NEB), 5U Klenow DNA polymerase (NEB) and 50U T4 PNK (NEB) in T4 DNA ligase buffer (Life Technologies) at 20deg for 30 min. cDNA was tailed with dATP using 15U Klenow (3’-5’ exo-) (NEB) in NEBuffer 2 at 37deg for 30 min, and 1:10 diluted Illumina adapters were ligated using a Ligafast kit (Promega) followed by gel purification of ~200bp species. Libraries were amplified for 15 cycles using Phusion polymerase (NEB) and Illumina PCR primers 1.0 and 2.0.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Description

processed data file: ORF analysis log quant for GEO.txt, 100bp quant for GEO.txt

Data processing

Quality and adapter trimming using Trim Galore (v0.3.5; default options; http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/)
Aligned to the yeast genome (build SGD1.01) using Bowtie (Langmead et al., 2009) (v0.12.7; default settings)
Genome_build: SGD1.01 (December 2006 build; http://may2009.archive.ensembl.org/Saccharomyces_cerevisiae/Info/Index)
Supplementary_files_format_and_content: ORF analysis log quant for GEO.txt - log2 transformed readcounts for wildtype and trf4 for each ORF. Chromosome and start and end positions are given for each entry.
Supplementary_files_format_and_content: 100bp quant for GEO.txt -The genome was divided into 100bp tiles and log2 transformed readcounts for wildtype and trf4 are given for each. Chromosome and start and end positions are given for each entry, along with any overalpping feature. This is the data used for CUT analysis in the associated publication.

Submission date

Aug 08, 2014

Last update date

May 15, 2019

Contact name

Jonathan Houseley

E-mail(s)

jon.houseley@babraham.ac.uk

Organization name

Babraham Institute

Department

Epigenetics Programme