GSM5734409: 20JV_Experiment3; Mus musculus; RNA-Seq
Single cells were index sorted into 384 well plates containing 1uL of reverse transcription (RT) master mix using a FACS Aria III with FACS Diva software (BD Biosciences). The RT master mix consisted of: 0.2μL SuperScript IV 5x buffer (Invitrogen), 0.03μL SuperScript IV taq (Invitrogen), 0.03 DTT 0.1M (Invitrogen), 0.03μL RNaseOUT (Invitrogen), 0.012μL dNTPs (Invitrogen), 0.19μL RNase Free H2O and 0.5μL of oligoDT primer [1μM stock] (custom made, IDT) per well. A unique oligoDT primer that contains a 6 basepair well barcode and a 10 54 basepair unique molecular identifier was added to each well. Reverse transcription was performed at 42C for 50 minutes followed by heat inactivation at 80C for 10 minutes. cDNA libraries were amplified using a custom targeted strategy the exact detailsf of which are described by Andrew G. Shuparski et. al. Single cell qtSEQ: cell-indexed quantitative and targeted RNA sequencing for sorted rare lymphocyte subpopulations STAR Protocols 2021