title: GSM5834690: HC-29 Day 1 (P3.H08); Homo sapiens; RNA-Seq
library_construction_protocol: Blood was drawn directly into the Tempus™ Blood RNA Tube (Thermo Fisher Scientific, Waltham, MA) according to manufacturer's protocol. Two Tempus tubes were collected at each study timepoint. The blood sample from each Tempus tube was aliquoted in to two 4.5mL cryovials (General lab supplier). These cryovials were directly stored at -80°C. RNA was isolated from tempus blood using the QIAsymphony RNA Kit (Qiagen, Gaithersburg, MD) on QIAsymphony SP instrument (Qiagen, Gaithersburg, MD). On the QIAsymphony, RNA CT 800 protocol was selected and used for RNA isolation. The instrument was set up according to the manufacturer's protocol and the elution volume for RNA samples was set to 100µl. The final volume of the eluted RNA samples ranged from 65 – 95 µl. RNA-seq libraries were prepared manually using Universal Plus mRNA-Seq with NuQuant, Human Globin AnyDeplete (Tecan Genomics, Redwood City, CA) according to manufacturer's protocol. For each sample, 500ng of total RNA was used to isolate mRNA via poly(A) selection. Captured mRNA was washed, fragmented, and primed with the mix of random and oligo(dT) primers. After cDNA synthesis, ends were repaired and ligated with Unique Dual Index (UDI) adaptor pairs. Unwanted abundant transcripts from rRNA, mtRNA and globin were removed using AnyDeplete module. Remaining library was amplified by 14 cycles of PCR and purified with AMPure XP reagent (Beckman Coulter, Indianapolis, IN).