title: GSM6736632: HEKNIH, dry ice; Homo sapiens; Mus musculus; RNA-Seq
library_construction_protocol: For each sample, scRNA was extracted from a mix of NIH and HEK cells (50%-50%; 10,000 cell input), 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. For the sample HEKNIH freezer and HEKNIH dry ice, the hydrogel with cell-bead tandems is frozen right after immersion to lysis buffer. This freezeing was done in -80°C freezer for the sample HEKNIH freezer, on dry ice for the sample HEKNIH dry ice. The day after, the frozen sample was thawed at 25°C for 15 min to resume cel lyeis step.  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 to obtain cell barocded cDNA. For each of the 3 samples, cDNA from 3 subfractions of PCR were individually purified with SPRIselect to remove short fragments. Sequencing libraries were prepared with illumina's Nextera XT kit for each of the 3 purified 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.