GSM8278772: BM-pDC, WT non-stim, rep1; Mus musculus; OTHER
RNA from BM-pDC was harvested using Qiagen Rneasy Plus Micro kit. RNA from primary splenic B cells was isolated using Zymoresearch Quick-RNA Miniprep kit. RNA libraries were prepared using BRB-Seq kit from Alithea Genomics, according to the manufacturer's recommendations. A total of 85 ng purified RNA was transferred into the BRB-Seq multiwell plate containing the barcoded oligo-dT primers for the reverse transcription step. All cDNA were pooled into an Eppendorf tube and purified using SPRIselect beads (Beckman) at a 1X ratio. The remaining free primers were digested with an exonuclease step. Second strand synthesis was performed from the purified cDNA pool and cleaned-up with a 0.6X SPRIselect beads step. Double stranded cDNA was then tagmented and purified with a 0.6X SPRIselect beads step. Finally the 3' terminal fragments were amplified using Unique Dual Indexing (UDI) Illumina adapter primers. After a final 0.7X SPRIselect beads purification step, the library generated was quantified with a fluorimetric method (QubIT, Thermo Scientifics) and its size pattern analyzed with a fragment analyzer (Agilent).