title: GSM5819424: Peripheral T cells of patinet #18 before TTF treatment; Homo sapiens; RNA-Seq
library_construction_protocol: Frozen PBMC samples were quickly taken from liquid nitrogen and thaw at 37 degree. T lymphcytes were isolated using Pan T Cell Isolation Kit, human (Miltenyi Cat#130-096-535), and the total RNA was extracted using Qiagen RNA extraction kit. RNA-seq library performed at ICBR Gene Expression & Genotyping (RRID:SCR_019145) using 25 ng of protein-free, DNase-treated total RNA (each sample processed individually) as input. Briefly, 25ng of total RNA was used for mRNA isolation using NEBNext Ploy(A) mRNA Magnetic Isolation module (New England Biolabs, catalog # E7490 ). Then followed by RNA library construction with NEBNext Ultra II Directional Lib Prep (New England Biolabs, catalog #E7760) according to the manufacturer's user guide. Briefly, RNA is fragmented in NEBNext First Strand Synthesis Buffer by heating at 94 °C for desired time. This step is followed by first strand cDNA synthesis using reverse transcriptase and oligo dT primers.  Synthesis of ds cDNA is performed using the 2nd strand master mix provided in the kit, followed by end-repair and adaptor ligation. Finally, library is enriched (each library has an unique barcode) by 12 cycles of amplification, and purified by Agencourt AMPure beads (Beckman Coulter, catalog # A63881).