title: GSM6736635: Pancreas islet, control, rep 1; Mus musculus; RNA-Seq
library_construction_protocol: For each replicate, scRNA was extracted from a 10,000 pancreas islet cells according to the RevGel-Seq protocol instructions. Pancreas islets were at fisrt isolated from whole pancreas by collagenase injection followed by handpicking under microscope. The islets were then dissociated with trypin-EDTA and the cell suspension was re-suspended in DPBS with 2% FBS and 0.5mM EDTA prior to following RevGel seq protocol. 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 are performed. Each sample is then split in 8 subfractions and PCR-amplified to obtain cell barocded cDNA. The amplified cDNA was pooled into three groups, 2 groups from 3  PCR tubes and 1 group of 2 PCR tubes, for purification with 0.6X SPRI select for subsequent sequencing library preparation. Sequencing libraries were prepared with illumina's Nextera XT kit for each of 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.