3'-DGE High Throughput RNA Library Preparation v2

This protocol is for the high-throughput transcriptome screening method called, 3’ Digital Gene Expression (3’-DGE). In this method RNA is purified from cell lysate and cDNA is generated by oligo dT priming, during which unique molecular identifiers (UMIs) are incorporated and strand-specificity is preserved. The 3’-DGE protocol has been optimized for 384 well plate experiments with a few thousand cells per well. The 3’-DGE method and a similar method known as DRUG-seq have been shown to be able to recapitulate 85% of the transcriptome you can obtain with standard mRNA-seq. In our experience using 3’-DGE we sequence each well at a low depth, aiming for ~1 million reads per sample which allows us to asses 40-80% of the transcriptome depending on the cell type. It is possible to sequence these samples to a greater depth if warranted. It is important to keep in mind that in 3’-DGE, and most low-input methods, only the 3’ poly-A end of the transcript is preserved. Therefore, this technique cannot be used if it is important to capture splice-isoforms or transcripts that do not have a poly-A tail. This is a modification of the SCRB-seq protocol originally published here: https://www.biorxiv.org/content/10.1101/003236v1; Recognition also goes to the Enard group and their modifications to SCRB-seq found here: https://www.protocols.io/view/mcscrb-seq-protocol-p9kdr4w The home-brew SPRI beads used in this study are made with this protocol: dx.doi.org/10.17504/protocols.io.bkppkvmn