GSM8098082: PBMCs from sample C8-7_Memory_CD8; Homo sapiens; RNA-Seq
RNA was purified from each sorted sample using the RNAeasy Plus Micro kit (Qiagen) Libraries were prepared for TCR sequencing as described by Rubelt, et al.. This was performed at the University of Cape Town. Briefly, purified RNA was reverse transcribed in a Rapid amplification of cDNA ends (RACE) reaction using SMARTScribe Reverse Transcriptase using previously designed oligos (isoC-5′-GTCAGATGTGTATAAGAGACAGnnnnnnnnnnCGATAGrGrGrG -3′-C3_Spacer and for the Poly A tail 5′-GTGTCACGTACAGAGTCATCtttttttttttttttttttttttttttttt -3′ VN) that captures polyadenylated transcripts and introduces a 10bp unique molecular identifier (UMI) at the 5' end of the product. cDNA was purified with AmpureXP beads (Beckman Coulter) before whole transcriptome amplification using Advantage 2 Polymerase (Clontech) using oligos that introduce Illumina Nextera Multiplex Identifier (MID) P5 Adapter sequences. The libraries were then sent to Stanford University (Department of Microbiology and Immunology, Stanford University) in a blinded fashion, where TCRβ-specific amplification was achieved with Q5 Hot Start Master Mix (NEB) using constant region-specific oligos, simultaneously introducing P7 MID sequences. Final sequencing libraries were purified using SPRISelect beads (Beckman Coulter), quantified using the Agilent TapeStation and pooled for sequencing. Paired-end sequencing was performed on an Illumina NovaSeq SP with 2x250 cycles, performed by the Chan-Zuckerberg Biohub Initiative.