title: GSM6736637: human PBMC control; Homo sapiens; RNA-Seq
library_construction_protocol: For human PBMC control, scRNA was extracted from 10,000 human PBMCs, according to the RevGel-Seq protocol instructions. Briefly, barcode beads and cells are attached via a bifunctional chemical linker. These tandems are dispersed in an hydrogel in the liquid state and immobilized upon gelation. Following cell lysis, polyA+ RNA molecules are captured on the barcoded beads via polyA-polyT interactions. Barcoded beads (and associated RNA molecules) are recovered following degelation, and reverse transcription and subsequent second strand synthesis with random primer are performed. Each sample is then split in 8 subfractions and PCR-amplified.  For the human PBMC 3x loading sample, following modifications were brought to the human PBMC control sample prep: the number of input cells is 30 000, all reagents quantity were doubled, PCR was made in 16 split tubes. For each sample, barcoded cDNA of 2 PCR tubes were pooled then purified with 0.6X SPRIselect to remove short fragments. Sequencing libraries were prepared with illumina's Nextera XT kit for the purified pooled barcoded cDNA according to manufacturer's instructions with the following adaptations: 600pg of the cDNA was subjected to tagmentation reaction. The tagmented DNA was amplified by 12 cycles PCR. During this PCR, DNA fragments with cell barcode sequence at one end are selectively amplified by using a primer pair of custom P5-read1 primer to the original barcode end and Nextera N7xx P7 index primer to the tagmented end. Paired end sequencing was done.