Total RNA was isolated using TRIzol Reagent, DNase-treated, and further purified using RNeasy mini spin columns (Qiagen). Small RNAs (>17 nt, < 200 nt) were purified using the mirVana miRNA isolation kit (Invitrogen). The 5.8S and 5S ribosomal RNAs were removed using the RiboMinus Eukaryote System v2 (Invitrogen). The purified small RNAs were treated with T4 Polynucleotide Kinase (NEB), purified using the Directzol microprep kit (Zymo) and their concentrations were determined using a Qubit 3 Fluorometer. TGIRT template-switching reverse transcription reactions and adapter ligations were performed as described previously (Qin et al., 2016; Shurtleff et al., 2017) using 50 ng of RNA. The ligated cDNA products were purified using a MinElute column (Qiagen) and PCR amplified using Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) and 200 nM of both Illumina multiplex primer and Illumina barcode primer (Table S6). PCR was performed for 17 cycles, as described previously (Shurtleff et al., 2017), and PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter).
GEO Accession
GSM4146709