title: GSM6283048: Xrn1 ko A549 cells, mock, biol rep 2; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was harvested using RNeasy Plus mini kit (Qiagen) and treated with Turbo DNase (Life Technologies). 3 ug of total DNase treated RNA was used for the construction of sequencing libraries. 3 µg RNA was mixed with 6 µl of ERCC ExFold RNA spike-in mixes diluted to 1:100 (Invitrogen). Ribosomal RNA was removed from each sample using Ribo-Zero Plus kit (Illumina). The rest of the RNA was ligated to a RACE-Seq adapter using T4 RNA ligase (Invitrogen) for 2 hours at 25C. The RACE-seq adapter contains a unique molecular identifier (UMI) in order to identify and remove reads that are the result of PCR duplication during the library preparation. Ligated RNA was then reverse transcribed to cDNA with SuperScript III RT (Invitrogen) using random primers of different lengths that also include a known sequence adapter. Finally, reads were amplified in two rounds of PCR of six cycles each to enrich for ligated fragments and add the Illumina adapters. Between the two PCRs, DNA fragments of 150-400 bp were selected using SPRIselect Reagent (Beckman Coulter).