GSM4768044: CT14_20190001_AR01; Drosophila melanogaster; RNA-Seq
The single cell library prep was based on CEL-seq2 with some modifications. Two rounds in vitro transcriptions were used to amplify ploly A mRNA. The RNA from the first round of IVT was purified by 0.8 fold RNAClean XP beads (0.8 fold ratio) and was used as a substrate for another round of first strand synthesis at 42 °C for 2 hours using second round primers (2nd Round Primer: NNNNNN), RNA:DNA hybrids were digested with RNase H (Thermal Fisher #18021014) at 37 °C for 30 minutes. The second round second strand synthesis was carried out at 16 °C with a T7-RA5 primer (GCCGGTAATACGACTCACTATAGGGAGTTCTACAGTCCGACGATC). The resulting cDNA underwent another final second round IVT step at 37 °C overnight and followed EXO-SAP treatment (Affymetrix 78200) for 15 minutes at 37 °C. Other steps were performed as described in the CEL-Seq2 protocol.