title: GSM7224145: WT, B17; Homo sapiens; OTHER
library_construction_protocol: ~42-48hrs post-transfection total RNA was extracted using home-made Trizol reagent (according to Chomczynski and Sacchi, 1987, 10.1016/0003-2697(87)90021-2), 1ml trizol was used per 1 well, following precipitation RNA was repurified following treatment with proteinase K at 20ng per microliter in a Tris-buffered 1% SDS solution with 2mM EDTA and 100mM NaCl, purified by phenol-chloroform extraction, then samples  were deprived of DNA by treatment with Tubro DNase (Ambion) and again purified by phenol-chloroform extraction For the 12 home-made 293T cell lines (this paper) including the wild-type and the XRN1 KO each biological replicate was a single technical replicate (one index per cell line). For the 4 acquired cell lines (10.1021/acs.biochem.0c00069) three technical replicates (starting at the RT step) of each biological replicate were performed and marked with a different index. Synthetic spike-ins had defined index sequences see Supplementary Table 1. Libraries were prepared as described in (10.1016/j.cell.2018.07.022) with modifications. Briefly, 2µg total RNA after DNase treatment were ligated with 100pmole RA3_15N 5' preadenylated adapter comprising a 15-nucleotide unique molecular identifier (UMI) in 20µl of 1x enzyme-supplied buffer, 15% PEG 8000 by T4 RNA ligase 2 truncated (NEB, M0242) for 18h at 18˚C. Nucleic acids of each reaction were purified using 20µl Ampure XP magnetic beads, eluted in and used for reverse transcription in 20µl with 100pmoles RPI primer (containing an 6-nucleotide index) using Superscript III (ThermoScientific) enzyme according to the manufacturer's protocol (2mins at 50˚C, 30mins at 45˚C, 15mins at 85˚C), followed by purification on 1,25 volume of Ampure XP magnetic beads. Nested PCR was performed with PrimeSTAR GLX polymerase (TaKaRa, R050B) since it proofed best at elongating long poly(A) tracts (as compared to Phusion of ThermoScientific and Q5 polymerases of NEB; not shown). First round PCR were performed in 25µl with an equivalent of 500ng input total RNA (input to RT step) per reaction. Primers at 0,35µM were either of: 3UNP1f, GAPDH_3R0, PABPC4_3R0 for L1, GAPDH, and PABPC4 respectively, universal reverse primer, RP_uni, was added at 0,4µM. Reactions were carried out: 98˚C 2mins, [98˚C 12sec, 52˚C 15sec, 70˚C 40sec]x21, 68˚C 1min, 1/5 of each reaction was used without purification as template for second round PCR with 0,36µM transcript-specific forward primer: L1_NGS4, or GAPDH_NGS, or PABPC4_NGS, and 0,4µM universal reverse primer RP_uni. PCRs were carried out: 98˚C 2mins, [98˚C 12sec, 55˚C 15sec, 70˚C 30sec]x21, 70˚C 1min. The reactions were purified on 1,3 volume Ampure XP magnetic beads, eluted, measured and diluted to ~5ng/µl, and analysed on TapeStation (Agilent). Before running on Illumina Novaseq libraries were quantitated by qPCR to adjust the number of reads to ~3-5 MRds for each transcript for each biological/technical replicate (each index).