Solid rna barcoding kit module 1 16

SAGE-seq libraries were generated from 5 μg of total RNA using SOLiD SAGE Kit with Barcoding Adaptor Module and multiplexed using SOLiD RNA Barcoding Kit Module 1-16 (Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. Briefly, total RNA was treated with DNase I, and then bound to Dynabeads Oligo(dT) EcoP magnetic beads for cDNA synthesis. The double-stranded cDNA synthesized on the beads was digested with Nla III and ligated to the adaptor containing an EcoP15I restriction enzyme recognition site. The adapter-ligated cDNA was digested with EcoP15I to release the tag containing the adaptor sequence and 27 bp of unique sequence from a single transcript. Each library was amplified in a PCR reaction by 8 cycles.

Bacterial lysis and RNA purification was performed using a FastPrep-24 vertical homogenizer (MP BioMedicals) and FastPrep Lysing Matrix B tubes following a previously described protocol (Davis et al., 2013 (link)). After DNase treatment, an aliquot of sample was analyzed for the presence of genomic DNA contamination by PCR. Ribosomal RNA was depleted from total RNA preparations using a Ribo-Zero rRNA Removal Kit following the manufacturer’s protocol (Illumina). RNA quantity and quality was determined using NanoDrop 2000 (Thermo Fisher) and Agilent 2100 BioAnalyzer (Agilent Technologies). Sequencing samples were prepared using the SOLiD^® Total RNA-Seq Kit (Life Technologies) with 100 ng ribosomal RNA-depleted and barcoded (SOLiD™ RNA Barcoding Kit, Module 1-16, Life Technologies) according to the manufacturer’s protocol. Emulsion PCR and SOLiD sequencing, 75 base pairs single direction, were performed according to manufacturer’s instructions for the SOLiD™ 5500 System (Life Technologies).