title: TGIRT-seq of A549: Ribo-depleted, Unfragmented
design: Total RNA from the indicated cell line was ribo-depleted by using a RiboZero Gold (human/mouse/rat) kit (Illumina). The resulting ribo? RNA was then either mock-treated or digested with 1 U of Terminator exonuclease (Epicentre). Pretreated RNAs were then incubated with T4PNK (Epicentre) to remove 3? phosphates or 2?,3? cyclic phosphates. Additionally, for fragmented libraries, a subset of pretreated RNAs was fragmented prior to T4PNK treatment with an NEBNext Magnesium RNA fragmentation module (New England Biolabs) by incubating RNAs for 7 min at 94°C. RNA reaction cleanup used a modified version of the Zymo RNA Clean and Concentrator (Zymo Research) protocol (i.e., addition of eight sample volumes of ethanol to increase retention of very small RNA species). cDNAs were synthesized via TGIRT template switching with 1 ľM purified TeI4c-?En-MRF reverse transcriptase for 15 min at 60°C. During synthesis, a DNA oligonucleotide containing the complement of an Illumina Read 2 sequencing primer-binding site was seamlessly linked to the 5? cDNA end. After reaction cleanup, a DNA oligonucleotide containing the complement of an Illumina Read 1 sequencing primer-binding site was then ligated to the cDNA 3? end by using thermostable 5? AppDNA/RNA ligase (New England Biolabs). Properly ligated cDNAs were then amplified for 12 cycles of PCR in order to synthesize the second strand and add Illumina capture and index sequences. Libraries were size-selected with Ampure XP beads (Beckman-Coulter) and evaluated on an Agilent 2100 Bioanalyzer.
identifiers.namespace: SUB1925895
identifiers.id: A549, ribo(-), +phos