sample B
2ug of Human Brain Reference RNA (Life Technologies) was ribo-depleted and fragmented to a median length of 110 nt by using an NEBNext RNA Fragmentation Kit (New England Biolabs) and treated with phage T4 polynucleotide kinase (Epicentre) to remove 3’ phosphates and 2’, 3’ cyclic phosphates, which impede TGIRT template switching. RNA-seq libraries were constructed from half of the recovered RNA used for cDNA synthesis viaby TGIRT template switching with 1 M TGIRT-III RT (InGex) for 15 min at 60o C, as described (Qin et al. 2015). The initial template-switching reaction seamlessly links an Illumina Read2 sequencing primer-binding site adaptor to the beginning of the cDNA sequence, after which an Illumina Read1 sequencing primer-binding site adaptor is ligated to the other end of the cDNA by using Thermostable 5’ AppDNA/RNA Ligase (New England Biolabs). The cDNAs with Read1 and Read2 sequencing adaptors on either end were then amplified for 12 cycles of PCR, during which Illumina multiplex and index sequences were added. Libraries were size-selected by using AmpureXP beads (Beckman-Coulter) and evaluated on the Agilent 2100 Bioanalyzer. TGIRT-seq libraries were sequenced using the Illumina NextSeq 500 platform (PE 2 x 75) at the Genome Sequencing and Analysis Facility (GSAF) at the University of Texas at Austin.