GSM5905243
MetOH is washed twice with ddw. Cells are subjected to proteinaseK treatment, followed by potassium-acetate precipiation. Supernatanant is passed through nucleic-acid binding column, washed multuple time in 60% EtOH and eluted in 10 mM TRIS  pH 7.5. Up to this point DTT is added to maintain reducing conditions. Finally, RNA is roughly quantified and subjected to an alkylation reaction (4tU->C), followed by another binding-washing-elution step. polyT primers with barcode and UMI are added to each sample and reverse transcription is performed. Samples are pooled, and subjected to a tn5 reaction. Finally an elongation step to extend transposase adapters and a PCR is performed to enrich tagged cDNA. In this protocol R2 starts with a 7bp sample barcode, followed by a 8 bp UMI followed by putative polyT. R1 immediately starts by reading the sense strand, enriched to the 3' of polyA mRNA. I2 is added in PCR and denotes a specific library/experiment. I1 is a constant sequence - ATCTCGTA.