alias: Bark
title: Bark
design: RNA sequencing libraries were prepared with Illumina-compatible NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, MA, USA) at Genotypic Technology Pvt. Ltd., Bangalore, India. 1 g of pooled total RNA was taken for mRNA isolation, fragmentation and priming. Fragmented and primed mRNA was further subjected to first strand synthesis in the presence of Actinomycin D (Gibco, life technologies, CA, USA) followed by second strand synthesis. The double stranded cDNA was purified using HighPrep magnetic beads (Magbio Genomics Inc, USA). Purified cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per NEBNext Ultra Directional RNA Library Prep Kit protocol. Adapter ligated cDNA was purified using HighPrep beads and was subjected to 12 cycles of Indexing-PCR (37 C for 15mins followed by denaturation at 98C for 30 sec, cycling (98C for 10sec, 65C for 75sec) and 65C for 5mins) to enrich the adapter-ligated fragments. Final PCR product (sequencing library) was purified with HighPrep beads, followed by library quality control check. Illumina-compatible sequencing library was quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA) and its fragment size distribution was analyzed on Agilent 2200 Tapestation.
identifiers.namespace: SUB4203357
identifiers.id: Bark