GSM4721919: PBS1_RNAseq; Mus musculus; RNA-Seq
We employed a modified version of the ribosome profiling protocol described by McGlincy et al. Flash-frozen embryonic brain tissue was thawed in 400 ml lysis buffer (150 mM NaCl, 20 mM Tris, pH 7.4, 5mM MgCl2, 1mM DTT, 100 mg/ml cyclohexamide, 1% v/v Triton X-100, and 25 U/ml Turbo DNase I (ThermoFisher Scientific)). Tissue was pipetted ten times in lysis buffer and incubated for 10 minutes on ice. Tissue was then triturated ten times through a 26-gauge needle. Lysate were clarified by centrifugation for 10 min at 20000 × g at 4°C and supernatant was recovered. Total RNA purified using the Direct-zol RNA MiniPrep kit (Zymo) with DNaseI treatment. RNA-seq libraries were prepared from 10 ng total RNA using the SMARTer® Stranded Total RNA-Seq Kit v2 (Takara) and sequenced on a Next-seq 500 using paired-end 2 x 37nt sequencing runs. For ribosome profiling, 1.5ml RNase I (10U/ml, ThermoFisher Scientific) was add to lysate containing 30 mg RNA and incubated for 45 minutes at room temperature with gentle agitation. 10 µl SUPERaseIn RNase inhibitor (20U/ml, ThermoFisher Scientific) was added after 45 minutes and samples transferred to ice to stop nuclease digestion. Lysate was transferred to a polycarbonate ultracentrifuge tube and underlayed with 0.9 ml sucrose cushion (1 M sucrose, 20 U/ml SUPERaseIn in Polysome buffer, 150 mM NaCl, 20 mM Tris, pH 7.4, 5mM MgCl2, 1mM DTT, 100 mg/ml cycohexamide). Ribosome-protected RNAs were pelleted by centrifugation at 52,000 rpm, 4°C for 2 h. Supernatant was removed, and pellet resuspended in 300 µl TRIzol reagent. RNA fragments were purified using a Direct-zol RNA MiniPrep kit (Zymo Research) and eluted in 100 µl RNase-free water. RNA was precipitated with one volume of isopropanol, 30 µg GlycoBlue, and 100 mM NaOAc pH 5.5 (300 µl total volume), and chilled at −20°C for at least 2 hours, or overnight. RNA was pelleted by centrifugation for 30 min at 20000 × g, 4°C, supernatant was removed, and pellets were air-dried for 10 min. Purified RNA was resuspended in 5 µl 10 mM Tris pH 8, and RNA 17-34 nucleotides was size selected using a 15% TBE-Urea gel (Life Technologies). RNA was extracted from gel sections in 400 ul RNA extraction buffer (300 mM NaOAc, pH 5.5, 1mM EDTA, 0.25% v/v SDS), then precipitated with 30 µg GlycoBlue and one volume of isopropanol. Precipitations were left at −20°C overnight. Size-selected RNA was resuspended in 4 µl 10 mM Tris pH 8 and transferred to a clean non-stick RNase-free microfuge tube. Size-selected RNA was dephosphorylated with T4 PNK (NEB) at 37°C for 1 hour. The reaction was brought up to 50 µl with nuclease-free water and purified using 1.8 volumes of Agencourt RNAClean XP beads (Beckman Coulter) and 5.4 volumes of isopropanol. Beads were captured on a magnetic rack and washed twice with 85% EtOH. Beads were air-dried and resuspended in 7 µl nuclease-free water. Supernatant containing purified RNA was removed from beads and quantified by Qubit RNA assay (ThermoFisher Scientific). Sequencing libraries prepared using the SMARTer® smRNA-Seq Kit for Illumina® (Takara). Libraries were sequenced on a Novaseq 6000 using single-end 1 x 50 nt sequencing runs.
GEO Accession
GSM4721919