RNA was prepared (RNEasy kit, QIAGEN) from total PBMCs and was
subjected to reverse transcription using barcoding primers that contain
unique Ab identifiers as previously described (Briney et al., 2016a (link)). The cDNA was then
amplified using a mix of gene specific primers. Illumina sequencing
adapters and sample-specific indexes were added during a second round of
PCR using 1μL of purified PCR product in 100μL of total
reaction volume and using the following thermal cycling program: 94C for
5 minutes; 10 cycles of 94C for 30 s, 55C for 30 s, 72C for 2 minutes;
72C for 7 minutes. Indexed PCR products were purified using 75μL
of SPRIselect beads (Beckman Coulter) and eluted in 50μL of
water. Samples were quantified using fluorometry (Qubit; Life
Technologies), pooled at approximately equimolar concentrations and the
sample pool was requantified. Samples were loaded onto an Illumina MiSeq
sequencer with a target loading concentration of 40pM and 10%
PhiX and sequenced (MiSeq 600-base v3 reagent kit; Illumina).
subjected to reverse transcription using barcoding primers that contain
unique Ab identifiers as previously described (Briney et al., 2016a (link)). The cDNA was then
amplified using a mix of gene specific primers. Illumina sequencing
adapters and sample-specific indexes were added during a second round of
PCR using 1μL of purified PCR product in 100μL of total
reaction volume and using the following thermal cycling program: 94C for
5 minutes; 10 cycles of 94C for 30 s, 55C for 30 s, 72C for 2 minutes;
72C for 7 minutes. Indexed PCR products were purified using 75μL
of SPRIselect beads (Beckman Coulter) and eluted in 50μL of
water. Samples were quantified using fluorometry (Qubit; Life
Technologies), pooled at approximately equimolar concentrations and the
sample pool was requantified. Samples were loaded onto an Illumina MiSeq
sequencer with a target loading concentration of 40pM and 10%
PhiX and sequenced (MiSeq 600-base v3 reagent kit; Illumina).