Library quantification kit for truseq

cDNA libraries of CD45⁺ Live PBMCs were generated using the Chromium Single Cell 3ʹ Reagent Kits v2 (10x Genomics) protocol targeting 5,000 cells in two separate wells. Briefly, single cells were isolated into oil emulsion droplets with barcoded gel beads and reverse transcriptase mix. cDNA was generated within these droplets, then the droplets were dissociated. cDNA was purified using DynaBeads MyOne Silane magnetic beads (ThermoFisher, #370002D). cDNA amplification was performed by PCR (10 cycles) using reagents within the Chromium Single Cell 3ʹ Reagent Kit v2 (10x Genomics). Amplified cDNA was purified using SPRIselect magnetic beads (Beckman Coulter). cDNA was enzymatically fragmented and size selected prior to library construction. Libraries were constructed by performing end repair, A-tailing, adaptor ligation, and PCR (12 cycles). Quality of the libraries was assessed by using Agilent 2200 TapeStation with High Sensitivity D5000 ScreenTape (Agilent). Quantity of libraries was assessed by performing digital droplet PCR (ddPCR) with Library Quantification Kit for Illumina TruSeq (BioRad, #1863040). Libraries were diluted to 2 nM and paired-end sequencing was performed on a HiSeq 2500 sequencer (Illumina). The final read depths for the two technical replicates that we sequenced were 77,049 reads/cell and 86,246 reads/cell, respectively.