GSM8147203: ST_DR_GFP_4; Mus musculus; RNA-Seq
Peripheral blood or lung cells were isolated as previously described and sorted into 30 ul of lysis/binding buffer (home-made or Life Technologies) and stored at -80°C. mRNA was then captured with Dynabeads oligo(dT) (Life Technologies) according to the manufacturer's guidelines. A bulk variation of MARSseq 77 was used for preparation of RNAseq libraries. Briefly, RNA was first reverse-transcribed with MARSseq barcoded RT primers with the Affinity Script kit (Agilent). Reverse transcription was analyzed by qRT-PCR and samples with a similar CT were pooled. Each pool was treated with Exonuclease I (NEB) for 30' at 37°C. Double-stranded DNA was generated with the NEB second strand synthesis kit at 16°C for 2h.), Subsequently, in vitro transcription was performed over-night with the T7 High Yield RNA polymerase IVT kit (NEB) for 37°C overnight. Following IVT, the DNA template was removed with Turbo DNase I (Ambion) at 37°C for 15'. Amplified RNA was fragmented by incubation at 70°C for 3' in Zn2+ RNA fragmentation reagent (Ambion). RNA was then ligated to the MARSseq ligation adaptor with T4 RNA Ligase I (NEB) at 22°C for 2h. Ligated product was reverse-transcribed using the Affinity Script RT enzyme (Agilent) and a primer complementary to the ligated product. The library was completed and amplified through a nested PCR reaction with P5_Rd1 and P7_Rd2 primers and PCR ready mix (Kappa Biosystems). Library concentrations was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument. RNAseq libraries were sequenced using the Illumina NextSeq 500.