GSM8068204: PUS1-/- CMC treated biol rep 6; Homo sapiens; OTHER
total RNA was extracted using 1ml TRIzol per 10cm dish following the manufacturers protocol. 200µg total RNA per replicate was used for the construction of sequencing libraries. Pseudo-seq libraries were prepared as described before (Carlile et al., 2015) with some adaptations. Poly-A RNA enrichment was performed with Poly(A)Purist™ MAG Kit (AM1922, Thermofisher) using 200 µg total RNA input. RNA was fragmented in 10 mM ZnCl2 for 55 s at 94 °C and quenched with 20 mM EDTA followed by ethanol precipitation. RNA was then either CMC treated (0.4 M, +CMC) or mock treated (-CMC) in BEU buffer for 45 min at 40 °C and 1000 rpm. After ethanol precipitation, unspecific CMC labelling was removed by incubating the RNA in sodium carbonate buffer for 2 h at 50 °C and 1000 rpm followed by ethanol precipitation. RNA ends were repaired with T4 PNK (M0201L, NEB) for 2 h at 37 °C, precipitated and size selected (120-140 nt) by Urea-PAGE. A 3′ adenylated adapter (5′-AppGATATCGTCAAGATCGGAAGAGCACACGTCTGAA-ddC-3′) was then ligated to the RNA fragments using T4 RNA ligase (M0373L, NEB) for 4 h at 22 °C followed by ethanol precipitation. The reverse transcription was performed with AMV RT (M5108, Promega) and RT primer: 5′-pRNAGATCGGAAGAGCGTCGTGTAGGGAAAGAG-iSp18-GTGACTGGAGTTCAGACGTGTGCTC-3′. Truncated cDNAs (110-180 nt) were size selected using Urea-PAGE. Gel purified cDNA was circularized using CircLigase ssDNA ligase II (CL9025K, Epicentre) and PCR amplified with forward primer (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCT*C-3′) and barcode reverse primer (NEBNext® Multiplex Oligos for Illumina®, Index Primers Set 1-4, NEB E7335S, E7500S, E7710S, E7730S). PCR products were PAGE purified and sequenced on an Illumina NovaSeq 6000 using an SP or S2 flow cell in SR75 mode to yield a total of 130 million reads per library.