title: GSM5365702: Cohort1_044X_0_TET; Homo sapiens; RNA-Seq
library_construction_protocol: PBMCs were isolated at the Babraham Institute by density gradient centrifugation and cryopreserved in liquid nitrogen. Before sorting, ~10-30 million PBMC cells were thawed in culture media and rested for 2 hours at 37 degrees C, after which influenza HLADR tetramers (TET)added along with a panel of cell surface antibodies. Live CD3+ CD4+ CD45RA- TET+ cells were sorted on a BD Aria Fusion sorter directly into 1x lysis buffer as per the SmartseqV4 Ultra low input RNA kit for sequencing from Takara Clontech. Cells from day 7 after vaccination (cTfhTet) were also sorted on the basis of expressing PD1 and CXCR5 markers. cDNA was prepared directly from cells (40-1000 max) lysed in 1x lysis buffer, and amplified acccording to the protocol for the SmartseqV4 Ultra low input RNA kit for sequencing from Takara Clontech. Between 16 and 18 PCR cycles were used for each sample, depending on cell input. Quality of cDNA was confirmed using an Agilent Bioanalyser. Libraries were generated from amplified cDNA using the NexteraXTDNA library preparation kit from Illumina, with nexteraXT indexes. Library quantitatation was performed using Agilent Bioanalyser, KAPA qPCR and Qubit High-sensitivity kits.