title: GSM4838073: Breast_n3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted using Qiagen's RNEasy minikit according to their protocol with on-column DNAse 1 digestion then evaluated on an Agilent Bioanalyser. 5 µg of the RNA sample, to which a 1ul spike-in of ERCC EXfold mix 1 was added (Thermofisher Scientific) were ribo-depleted by using a RiboZero Gold (Human/Mouse/Rat) kit (Illumina). 50ng of the resulting sample were then fragmented using an NEBNext Magnesium RNA Fragmentation Module (New England Biolabs) at 94°C for 2 or 3min (depending on RIN) and treated with T4 polynucleotide kinase (Epicentre) to remove 3′ phosphates and 2′, 3′ cyclic monophosphates, which impede TGIRT template switching (Mohr et al. 2013). RNAs were purified after ribo-depletion, fragmentation, and dephosphorylation by using a modified version of the Zymo RNA Clean & Concentrator protocol (addition of eight sample volumes of ethanol to increase retention of very small RNA species). The recovered RNA was used for cDNA synthesis via TGIRT template switching with 1 μM TGIRT-III RT (InGex, LLC) for 15 min at 60°C, as previously described (Qin et al. 2016). The template-switching reaction seamlessly links the complement of an Illumina Read 2 sequencing primer-binding site (R2R DNA) to the 5′ end of the cDNA during cDNA synthesis, after which a DNA oligonucleotide containing the complement of an Illumina Read 1 sequencing primer-binding site (R1R DNA) is ligated to the cDNA 3′ end by using Thermostable 5′ AppDNA/RNA Ligase (New England Biolabs). Ligated cDNAs with R1R and R2R sequencing adapters on either end were then amplified for 12 cycles of PCR (Qin et al. 2016; initial denaturation at 98°C for 5 sec, followed by cycles of 98°C for 5 sec, 65°C for 10 sec, 72°C for 10 sec), during which Illumina capture and index sequences were added. Libraries were size-selected by using Ampure XP beads (Beckman-Coulter), evaluated on an Agilent 2100 Bioanalyzer, and quantified using Qubit 2.0 fluorometer (Invitrogen) as was the final equimolar pool of the TGIRT-seq libraries. They were then sequenced using the Illumina NextSeq 500 instrument (75-nt paired-end reads) with the Illumina NextSeq 500/550 High Output Kit v2 (150 Cycles).