GSM9380526: PBMCs, 3 timepoint samples, Adenovirus infection, BCR; Homo sapiens; RNA-Seq
PBMC isolation from fresh whole blood using SepMate™ tubes and STEMCELL Technologies Lymphoprep™ protocol. PBMCs were washed twice using PBS with 2% FBS then counted Eosin stain for viability. Cells were then centrifuged at 400 g for 5 minutes and resuspended in FBS with 10% DMSO for cryopreservation. For BD Rhapsody proccessing, cryopreserved PBMCs from the three timepoints were thawed simultaneously for loading onto a single-lane BD Rhapsody Cartridge and cell capture using Enhanced Cell Capture Beads V1 (BD Biosciences, cat# 664887). Cells were thawed in a 37°C water bath, washed in DMEM with 2% FBS Cell preparation, loading, lysis, and Capture Bead retreival were conducted as per manufacturer's protocol. All centrifugation steps were conducted at 400 x g for 5 minutes at 4°C. Library preparation was performed in accordance with BD Rhapsody Human TCR/BCR + WTA + Sample Tag Library Preparation Protocol. AMPure® XP magnetic beads were used for all PCR cleanups (Beckman Coulter, cat# A63880) and libraries were dual indexed with Illumina TruSeq and Nextera primers. Quality check of the dual indexed libraries was done using the Invitrogen Qubit™ dsDNA HS Kit and the Agilent High Sensitivity D5000 Tapestation kit. Libraries were pooled and sequenced on the Illumina NovaSeq X 25B flow cell with run configuration paired end 150 bp reads and loading concentration 100mM