GSM9262025: 40S_30nM_a; Saccharomyces cerevisiae BY4741; RNA-Seq
The frozen cell pellet from a 500 ml culture was thawed on ice and mechanically disrupted by vortexing with glass beads three times for 2 min each at maximum speed in a cold room (4°C) in 2 ml of buffer RL (provided in the kit), 3 ml phenol:chloroform:isoamyl alcohol 25:24:1 (pH 5.2) and 3 ml ice cold glass beads. Following 5 min centrifugation at 14000 rpm in a Sorvall LYNX 6000 Superspeed centrifuge using a Fiberlite F14-14 x 50cy rotor, the supernatant was transferred to a fresh tube and total RNA was prepared using a GenElute total RNA purification Maxi kit (Sigma-Aldrich; RNB200) following the manufacturer's protocol. 400 μg total RNA in 250 μl water was applied to a GeneElute mRNA miniprep kit (Sigma-Aldrich; MRN70-1KT), following the manufacturer's protocol. The purified total mRNA was treated by 5'-Phosphate-Dependent Exonuclease (Lucigen, TER51020) to degrade RNAs with 5' monophosphates, such as 18S and 25S ribosomal RNAs, at 0.3 μg/µl mRNA in 1X buffer A (provided in the kit), 1U/µl RiboGuard RNase Inhibitor (Lucigen; RG90925), 0.1 U/µl 5'-Phosphate-Dependent Exonuclease. Following a 1 h incubation at 30°C, the total mRNA was extracted using phenol:chloroform:isoamyl alcohol 25:24:1 (pH 5.2), ethanol precipitated, resuspended in 10 mM Tris (pH 8.0) and stored in -80 °C. RecSeq sequencing library construction was conducted according to a previously described protocol (McGlincy and Ingolia 2017), with modifications, using identical barcoded linkers (NI-810 to NI-815), RT primer (NI-802) and PCR primers (NI-798, NI-799, NI-822 to NI-824). The RNA fragments purified from 48S PIC RPFs were dephosphorylated using T4 Polynucleotide Kinase (PNK) (NEB; M0201L) and ligated to pre-adenylated linkers (NI-810 to NI-815) containing 5 nt sample barcodes unique for each sample using truncated T4 RNA ligase 2 (K227Q) (NEB; M0351L). Ligated fragments were separated from free linkers on a 15% polyacrylamide TBE-Urea gel and then pooled and purified for reverse transcription using RT primer NI-802 and ProtoScript II Reverse Transcriptase (NEB; M0368S). The ~105 nt cDNAs were separated from free RT primers on a 15% TBE-Urea gel and circularized using CircLigaseII ssDNA Ligase (Biosearch Technologies; CL9021K). PCR was carried out using forward primer NI-798, and reverse primers (NI-799, NI-822 to 824) as already described (McGlincy and Ingolia 2017) .