title: GSM8974601: YA myobudles, control, Day 21 replicate 1; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was harvested using Quiagen kit. Illumina TruSeq RNA Sample Prep Kit (Cat#) was used with 0.5 ug of total RNA for the construction of sequencing libraries. Sample quality was assessed using the Agilent TapeStation 4200 (Agilent Technologies, Inc). 250 ng of total RNA was used for library construction using NEB Ultra II Directional RNA-seq library preparation kit for Illumina according to manufacturer's protocol. First, 100 ng of total RNA was used for mRNA isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, catalog # E7490). Then followed by RNA library construction with the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, catalog #E7760) according to the manufacturer's user guide. Briefly, RNA was fragmented in NEBNext First Strand Synthesis Buffer via incubation at 94 °C for the desired time. This step was followed by first-strand cDNA synthesis using reverse transcriptase and random hexamer primer.  Synthesis of ds-cDNA was performed using the 2nd strand master mix provided in the kit, followed by end-repair and adaptor ligation.  At this point, Illumina adaptors were ligated to the sample.  Finally, each library (uniquely barcoded) was enriched by 12 cycles of amplification, and purified with Agencourt AMPure beads (Beckman Coulter, catalog # A63881). 15 barcoded libraries were sized on the TapeStation 4200 and quantified with the Qubit® 2.0 Fluorometer. Finally, Barcoded libraries were pooled equimolarly for sequencing simultaneously for NovaSeq