PBMCs from lupus patients were isolated and prepared as described above. Once resuspended in EasySep buffer, the EasyEights Magnet was used to sequentially isolate CD14+ (using the EasySep Human CD14 positive selection kit II, cat #17858), CD19+ (using the EasySep Human CD19 positive selection kit II, cat #17854), CD8+ (EasySep Human CD8 positive selection kitII, cat#17853), and CD4+ cells (EasySep Human CD4 T cell negative isolation kit (cat #17952) according to the kit protocol. RNA was extracted using the RNeasy Mini kit (#74104), and reverse transcription and tagmentation were conducted according to Picelli et al. using the SmartSeq2 protocol51, 52. After cDNA synthesis and tagmentation, the library was amplified with the Nextera XT DNA Sample Preparation Kit (#FC-131-1096) according to protocol, starting with 0.2ng of cDNA. Samples were then sequenced on one lane of the Illumina Hiseq4000 with paired end 100bp read length, yielding 350M total reads.
