T7 highscribe kit

In-vitro transcribed (IVT) RNA or purified Schizosaccharomyces pombe total RNA was used as spike-ins where noted. The IVT RNA was produced by first amplifying a linear DNA fragment encoding NanoLuc using Q5 polymerase (NEB #M0494S), and purifying the DNA using an NEB clean and concentrate kit. The RNA was then made using a T7 Highscribe kit (NEB #E2040S), treated with DNase I (NEB #M0303L) and purified using an NEB clean and concentrate kit (NEB #T2030).
For the S. pombe RNA, fission yeast (FY527) was grown in YES media (5 g/L yeast extract, 30 g/L glucose, 225 mg/L adenine, histidine, leucine, uracil and lysine hydrochloride) at 32°C until OD₆₀₀ = 0.5, harvested by centrifugation (3 minutes at 2500 g), resuspended in Trizol, and lysed by vortexing with 0.5 mm zirconia glass beads before extracting RNA using Zymo Direct-zol kits (Zymo #R2072).

In-vitro transcribed (IVT) RNA or purified Schizosaccharomyces pombe total RNA was used as spike-ins where noted. The IVT RNA was produced by first amplifying a linear DNA fragment encoding NanoLuc using Q5 polymerase (NEB #M0494S), and purifying the DNA using an NEB clean and concentrate kit. The RNA was then made using a T7 Highscribe kit (NEB #E2040S), treated with DNase I (NEB #M0303L) and purified using an NEB clean and concentrate kit (NEB #T2030).
For the S. pombe RNA, fission yeast (FY527) was grown in YES media (5 g/L yeast extract, 30 g/L glucose, 225 mg/L adenine, histidine, leucine, uracil and lysine hydrochloride) at 32°C until OD₆₀₀ = 0.5, harvested by centrifugation (3 minutes at 2500 g), resuspended in Trizol, and lysed by vortexing with 0.5 mm zirconia glass beads before extracting RNA using Zymo Direct-zol kits (Zymo #R2072).

To generate the HheCas13a and TccCas13a
gRNAs, 10 μM T7 promoter (oligo from IDT) was annealed to the
bottom strand, which contains the repeat region and the spacer for
the gRNA (single-stranded oligo). Annealing occurred in a PCR buffer
with a gradual decrease of temperature from 95 to 25 °C, in 5
°C decrements every 2 min. The annealed product was in vitro
transcribed (IVT) with a T7 highscribe kit (NEB) according to the
manufacturer’s protocol. The RNA product was purified with
zymo RNA purification kit and measured with a NanoDrop spectrophotometer.
To generate the AapCas12b, BrCas12b, and eBrCas12b gRNAs, the repeat
region was used as a single-stranded DNA ultramer (IDT). To make a
full-length DNA for the gRNA (containing the repeat and the spacer),
the spacer was used as a reverse primer (having a shared region with
the repeat region) and PCR was amplified with the forward primer T7
oligo. The PCR product was purified with Qiagen and the product was
measured with a NanoDrop spectrophotometer. Then, 300–1000
ng of the PCR product was used as a template for the IVT using Hiscribe
kit (NEB). The product was purified with a zymo RNA purification kit
and measured with a NanoDrop spectrophotometer.