GSM4984167: BR.e1; Homo sapiens; RNA-Seq
pDCs were detached from plates by gentle pipetting and stained for viability with the Zombie Aqua Fixable Vaiability Kit in PBS for 20 mins at 4C. Cells were washed in 4C medium and FAC-sorted to remove MRC5 cells based on forward and side-scatter using an SH800Z sorter (SONY Biotechnology). Total RNA from sorted live pDCs was isolated using an RNeasy Mini Kit (QIAGEN). Pooled RNA-seq was performed using the low-input RNA-seq protocol reported in Snyder et al, Science Immunology, 2019. All of the steps prior below that  come before the barcoded cDNA libraries are pooled were performed using a BioMek  4000 automated liquid handling robot (Beckman). Briefly, we added deoxyribonucleotides  (dNTPs) (ThermoFisher) to a final concentration of 2 mM and reverse transcriptase primer  to 2 uM followed by primer annealing at 72C for 3 mins. We then added 7.5 uL of reverse  transcription mixture to each well comprised of 2M betaine (Affymetrix), 2x ProtoScript  Buffer (NEB), 12 mM MgCl2 (ThermoFisher), 10 mM dithiothreitol (ThermoFisher), 5.3 U  of Protoscript II reverse transcriptase (NEB), 0.53 U of SUPERaseIN (ThermoFisher), and  2 uM template-switching oligo. The reverse transcription reaction was incubated at 42C  for 90 mins followed by 10 cycles of 50C for 2 mins, 42C for 2 mins, and 70C for 10 mins.  Next, 2 uL of exonuclease I (ThermoFisher) was added to each well (1.875 U of  exonuclease I in water) and the resulting mixture was incubated at 37C for 30 mins, 85C  for 15 mins, and 75C for 30s.