NextSeq 500 sequencing; Bulk RNA Barcoding and sequencing (BRB-seq) protocol for RNA-seq of 60 human LCL from the 1000G project
Bulk RNA Barcoding and sequencing (BRB-seq) protocol for RNA-seq of 60 human LCL from the 1000G project
EVB transformed lymphoblastoid cell lines (LCLs) directly from Coriell Cell Repositories (GBR, FIN, TSI) or originally from Coriell but grown at the University of Geneva (CEU, YRI) were shipped to ECACC (European Collection of Cell Cultures) as live cultures, in batches of ~30 samples from Coriell (GBR/FIN/TSI somewhat randomized) and 2 x ~90 samples (by population) from Geneva. In ECACC, these cell lines were cultured to approximately 1.2 x 10e8 cells. These cultures were split to produce 8 x cell banks of the samples, and a snap frozen pellet of 2 x 10e7 cells from a proliferating culture. The cell pellets were shipped from ECACC to University of Geneva in three batches, the first batch consisting of CEU/GBR/FIN/TSI samples, and the second and third batch with YRI and the rest of CEU samples. RNA was extracted in Geneva about 14 samples at a time, first extracting 2/3 of the first shipping batch with full randomization, then adding the second batch and randomizing among that and the remaining 1/3 of the first batch, and finally extracting the third batch. Total RNA was extracted from cell pellets using the TRIzol Reagent (Ambion). The pellets had been frozen at ECACC without any additives like RNAlater or TRIzol. In Geneva they were thawed, 1mL of TRIzol was added in each sample, and the samples were transferred to eppendorf tubes. The rest of the protocol followed the manufacturer's guidelines. No DNAse treatment was done to the RNA samples. RNA quality was assessed by Agilent Bioanalyzer RNA 6000 Nano Kit according to the manufacturer's instructions. RNA quantity was measured by Qubit 2.0 (Invitrogen) using the RNA Broad range kit according to the manufacturer's instructions. BRB-seq novel protocol (see referenced paper)
Experimental Factor: cell line
HG00118