title: GSM8624521: B_18_C; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was extracted from frozen tissue samples by homogenization in trizol followed by chloroform separation and extraction in an RNAeasy mini column (Qiagen Inc., Germantown, MD) prior to cleaning using the RNase-Free DNaseSet (Qiagen Inc., Germantown, MD). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification(Figure A). For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification(Figure B). The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.