RNA-seq for sf3b-1 alleles

RNA-seq for sf3b-1 alleles To study the transcriptional consequences of the K718E mutation, we used two homozygous mutant lines (CER218 and CER220) derived from two independent CRISPR/Cas9 events (cer3 and cer7 alleles, respectively), and the corresponding wild-type siblings (CER217 and CER221). Synchronized L4 animals were collected in M9 after growing for 41 hours at 20oC and total RNA was isolated using TRI Reagent and the PureLink® RNA Mini Kit, following the manufacturer’s instructions. sftb-1(cer6) homozygous larvae were obtained by automatically sorting non-red animals from a synchronized CER191 population grown at 20C for 23 hours, using a the COPAS worm sorter system.Two CER276 (cer39 allele) biological replicates were collected independently without sorting, after growing for 23 hours at 20C. Total RNA from the six samples was extracted using TRI Reagent, followed by aqueous and organic phase separation using 1-Bromo-3-chloropropane, and RNA was precipitated with 2 propanol. After treatment with TURBO DNase, RNA was purified following a phenol/chloroform extraction protocol. In all cases, total RNA was quantified by Qubit® RNA BR Assay kit (Thermo Fisher Scientific) and the integrity was checked by using RNA 6000 Nano Bioanalyzer 2100 Assay (Agilent).