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Sample GSM1252351

Query DataSets for GSM1252351

Status

Public on Nov 22, 2013

Title

EarlyMeiosisIME4plus15min_input

Sample type

SRA

Source name

Time Course Early MeiosisIME4plus15min input

Organism

Saccharomyces cerevisiae

Characteristics

strain: sk1
strain info: SAy995
antibody: none

Growth protocol

To induce synchronous meiotic entry, cells were pre-selected on 1% yeast extract, 2% peptone, 3% glycerol, 2% agar for 24 hours at 30°C, grown for 24 hr in 1% yeast extract, 2% peptone, 4% dextrose at 30°C, diluted in BYTA (1% yeast extract, 2% tryptone, 1% potassium acetate, 50 mM potassium phthalate) to OD600 = 0.2 and grown for another 16 hr at 30°C, 300 rpm. Cells were then washed once with water and re-suspended in SPO (0.3% potassium acetate) at OD600 = 2.0 and incubated at 30°C at 190 rpm. Cells were isolated from SPO at the indicated times, collected by centrifugation, re-suspended in pre-warmed 1% yeast extract, 2% peptone, 2% dextrose and incubated at 30°C at 190 rpm. For ectopic expression of the MIS complex, cells were collected after 150 minutes of mitotic growth in the presence of cupric sulfate, induced with 100 µM CuSO4 in rich complete synthetic media (2% glucose).

Extracted molecule

polyA RNA

Extraction protocol

RNA was extracted from cells using a standard hot acid phenol protocol. Briefly, cell pellets were resuspended in equal volumes of acid phenol:chloroform 5:1 pH 4.3-4.7 (Sigma), buffer AE (50 mM sodium acetate, 10mM EDTA 1% SDS) and glass beads. This mixture was vortexed for 15 minutes, followed by a 15 minute incubation at 65°C. Samples were centrifuged for 10 minutes (12,000g, 4°C); the supernatant isolated, re-extracted with phenol:chloroform::5:1, and precipitated with sodium acetate and isopropanol. Enrichment of polyadenylated RNA (polyA+ RNA) from total RNA was performed using Oligo(dT) dynabeads (Invitrogen) according to the manufacturer’s protocol. The mRNA was chemically fragmented into ~80-nt-long fragments using RNA fragmentation reagent (Ambion). The sample was then subjected to Turbo DNAse treatment (Ambion), followed by a phenol-chloroform extraction, and resuspension in 20 μl of IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5). 25 μl of protein-G magnetic beads were washed and resuspended in 200 μl of IPP buffer, and tumbled with 3 μl of affinity purified anti-m6A polyclonal antibody (Synaptic Systems) at room temperature for 30 minutes. Following 2 washes in IPP buffer, RNA was added to the antibody-bead mixture, and incubated for 2 h at 4°C. The RNA was then washed twice in 200 μl of IPP buffer, twice in low-salt IPP buffer (50 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5), and twice in high-salt IPP buffer (500 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5), and eluted in 30 μl RLT (Qiagen). To purify the RNA, 20 μl MyOne Silane Dynabeads (Life Technologies) were washed in 100 μl RLT, resuspended in 30 μl RLT, and added to the eluted RNA. 60 μl 100% ethanol was added to the mixture, the mixture attached to the magnet and the supernantant discarded. Following two washes in 100 μl of 70% ethanol, the RNA was eluted from the beads in 160 μl IPP buffer. Eluted RNA was subjected to an additional round of IP, by re-incubating it with protein-A magnetic beads coupled to anti-m6A antibody, followed by washes, elution from the protein-A beads and purification as above, followed by elution from the MyOne silane dynabeads in 10 μl H20.
Strand-specific m6A RNA-seq libraries were generated as described in (Engreitz et al., 2013). Briefly, RNA was first subjected to FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific), followed by a 3’ ligation of an RNA adapter using T4 ligase (New England Biolabs). Ligated RNA was reverse transcribed using AffinityScript Multiple Temperature Reverse Transcriptase (Agilent), and the cDNA was subjected to a 3’ ligation with a second adapter using T4 ligase. The single-stranded cDNA product was then amplified for 9-14 cycles in a PCR reaction. Libraries were sequenced on Illumina Miseq, HiSeq 2000 and/or HiSeq 2500 platforms generating paired end reads (25 or 30 bp from each end, depending on the platform).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Reads were initially mapped against all SK1 ribosomal RNA (rRNA) sequences using Bowtie (version 0.12.7), and all reads aligning to the rRNA were discarded. All remaining reads were aligned against the SK1 genome using Tophat (version 1.4.1). Parameters used were ‘--max-multihits 1 –prefilter-multihits’ and ‘–transcriptome-index’, for which we assigned a pre-indexed version of the SK1 transcriptome. An in-house script was then used to cast all reads aligning to the genome upon the SK1 transcriptome. Only reads fully matching a transcript structure, as defined by the transcriptome annotation, were retained. Such reads were computationally extended in transcriptome space from the beginning of the first read to the end of its mate, and coverage in transcriptome-space was calculated for each nucleotide across all transcripts.
Peak detection within genes. To search for enriched peaks in the m6A IP samples, we scanned each gene using sliding windows of 100 nucleotides with 50 nucleotides overlap. Each window was assigned a score, corresponding to the fold of the mean coverage in the window over the median coverage across the gene. Windows with scores greater than 4 (i.e. greater than 4-fold enrichment) and with a mean coverage >10 reads were retained. Overlapping windows were merged together, and for each disjoint set of windows in transcriptome space we recorded its start, end, and peak position (corresponding to the position with the maximal coverage across the window), and its peak score (corresponding to the fold-change of enrichment in coverage over the median gene level).
Ensuring that peaks were absent in input. We repeated the same steps for the input sample. We eliminated from all subsequent analysis all windows that were detected in both step 1 and in step 2.
Comparison of multiple samples. To identify peaks that were robustly present across multiple replicates of wild-type samples but absent in the ime4∆/∆ negative control samples, we applied the following strategy. We first merged the coordinates of all windows from all samples passing step 1 and 2, to define a set of disjoint windows passing these filters in at least one of the samples. For each such window, we recalculated the peak start, end, and peak position and peak score across each of the samples using the approach in step 1. To identify IME4 dependent peaks in S. cerevisiae, we required that (i) the peak is detected in at least two of three replicates, (ii) the distribution of peak scores across the wild-type samples is significantly different from its counterpart in the ime4∆/∆ samples (Student’s t-test, P value < 0.05), and (iii) the mean peak score across the WT replicates is at least 3-fold greater than the mean peak score in the ime4∆/∆ samples.
Genome_build: yeast sk1
Supplementary_files_format_and_content: bed

Submission date

Oct 28, 2013

Last update date

May 15, 2019

Contact name

Schraga Schwartz

Organization name

WEIZMANN INSTITUTE OF SCIENCE

Street address

Herzl 234, Department of Molecular Genetics

City

Rehovot