title: GSM4138604: shNC_2; Homo sapiens; RNA-Seq
library_construction_protocol: For PBMCs, whole blood was lysed in four volume of RBCs lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA, pH 7.4) at room temperature (RT) and erythrocytes were removed by washing with phosphate buffered saline (PBS). Leukocytes thus obtained lysed in TRI reagent (Sigma-Aldrich, St Louis, MO) for RNA isolation as per the manufacturer's protocol. Total RNA was treated with RQ1 DNase (promega) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260 nm/280 nm (A260/A280) using smartspec plus (BioRad). RNA integrity was further verified by 1.5% Agrose gel electrophoresis. For each sample, 10 μg of total RNA was used for RNA-seq library preparation. Polyadenylated mRNAs were purified and concentrated with oligo(dT)-conjugated magnetic beads (invitrogen) before used for directional RNA-seq library preparation. Purified mRNAs were iron fragmented at 95 ℃ followed by end repair and 5' adaptor ligation. Then reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomizedhexamer.
attributes.tag: GEO Accession
attributes.value: GSM4138604