GSM1252349: EarlyMeiosisIME4plus0min_input; Saccharomyces cerevisiae; RNA-Seq
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).
GEO Accession
GSM1252349