title: GSM4760601: Comp_CB839_ox_rep1; Mus musculus; OTHER
library_construction_protocol: Cells were lysed with cold TRIzol (15596018, Life Technologies) on ice. Lysates were shaken with chloroform 5:1, centrifuged at 18,600g and precipitated with 2.7x volumes of cold ethanol in presence of 30 μg of GlycoBlue Coprecipitant (AM9515, ThermoFisher) overnight. Samples were resuspended in 0.3M acetate buffer (pH = 4.5) with 10 mM EDTA and precipitated again. Next day, samples were resuspended in 10 mM acetate buffer with 1 mM EDTA. 2 ug of each RNA sample was treated with 10 mM of either sodium periodate (“oxidized sample”) or sodium chloride (“non-oxidized sample”) and incubated for 20 min at room temperature in the dark. Reactions were quenched with glucose for 15 min. Yeast tRNAPhe (R4018, Sigma-Aldrich) was added to each sample, after which samples were precipitated with ethanol. Samples were resuspended in 50 mM Tris buffer (pH=9) and incubated for 50 min at 37C, quenched with acetate buffer and precipitated Samples were resuspended in RNAse-free water and subjected to a ligation to a 5'-adenylated DNA adaptor (5′-/5rApp/TGGAATTCTCGGGTGCCAAGG/3ddC/-3′), using truncated KQ mutant T4 RNA ligase 2 (M0373, New England Biolabs), for 3 hours at room temperature. Reverse transcription was performed with SuperScript IV reverse transcriptase (18090050, Thermo Scientific) according to the manufacturer's instructions, with a primer complementary to the DNA adaptor. After the reverse transcription step, 3' end of cDNA was ligated to a 5'-phosphorylated DNA adaptor (5'-/5phos/AGATCGGAAGAGCGTCGTGTAGGGA/3ddC/-3') using T4 RNA ligase 1 (M0437, New England Biolabs) in presence of 20% PEG, 1 mM ATP and 30 mM hexammine cobalt(III) chloride (H7891, Sigma-Aldrich). Ligation products were subjected to 8 cycles of PCR with primers containing 8-mer barcodes and p5 and p7 Illumina adaptors. PCR products were run out on an agarose gel and ~190 bp bands were excised, purified and subjected to MiSeq paired-end Illumina sequencing.