FL-cDNA Nanopore sequencing of HAP1-dEGFP: LbuCas13a with dEGFP crRNA , 50 minutes post transduction, rep 3
crRNAs were prepared by in vitro transcription. LbuCas13a protein was expressed in E. coli and purified in house. Cells were transduced using iTOP (D'Astolfo et al. 2015, Cell). RNA was harvested in TRIzol and purified using the zymo RNA clean and concentration -5 kit, including an in column DNase treatment. Next, ribosomal RNA was depleted with the RiboMinus Eukaryotic system v2. rRNA depleted total RNA was first poly(A) tailed with E.coli poly(A) polymerase in the presence of 1 mM ATP and 1 unit/uL RNasin (Promega, N2515) for 30 minutes at 37oC. Afterwards, the RNA was cleaned with the Zymo RNA Clean & Concentrator -5 Kit (R1013). Next, reverse transcription was performed using SuperScript IV RT (Invitrogen, 18090010) and anchored oligo dT20 primers. To remove RNA before second strand synthesis, 1 uL of RNase Cocktail (Thermo, AM2286) was added after reverse transcription and incubated at 37oC for 20 minutes. For the second strand synthesis, 1 L of 100 ng/L random hexamer primers (Promega: C1181) was added to each sample. Then, the NEBNext second strand synthesis kit (NEB, E6111) was used according to the manufacturers instructions. Finally, native barcodes (EXP-NBD104 & EXP-NBD114) and sequencing adapters (SQK-LSK109) were ligated.