Purification of RNA from sorted cells using Dynabeads mRNA DIRECT Purification Kit We barcoded RNA from each sample in a retrotranscription (RT) reaction with AffinityScript Multiple Temperature Reverse Transcriptase (Agilent, Santa Clara, CA) and different RT primers. After qPCR, cDNA with similar Ct values were pooled together. cDNA was purified with SPRIselect 1.2X (Beckman Coulter –BC-, Brea, CA) and amplified using the T7 promotor as template previously introduced in the RT reaction. T7 polimerase (NEB) was added for 16 hours at 37ºC. RNA molecules were fragmented with 2µL of 10X Zn2+ fragmentation buffer (Ambion™, ThermoFisher) for 1 min at 70ºC and purified with SPRIselect 2X. Afterwards, a ssRNA adaptor (Illumina, San Diego, CA) was ligated to the 3'-end of the RNA fragments in presence of DMSO, 100 mM ATP, 50% PEG and T4 RNA ligase I (NEB) for 2 hours at 22ºC. A second RT reaction was performed with AffinityScript Multiple Temperature Reverse Transcriptase and resulting cDNA was purified with SPRIselect 1.5X. Finally, cDNA was amplified with 12.5µL Kappa Hifi ready mix + 1µL 25 µM primer mix per sample and purified with SPRIselect 0.7X. Qubit, TapeStation and qPCR analysis were done as quality controls.
GEO Accession
GSM4520752