Dna free dnase treatment kit

We performed RNA-seq of five replicates (20 heads per replicate obtained from five independent matings) for each of WT and homozygous fam50a^−/− maintained on a WT background. At 2 dpf, we decapitated larvae and subjected tails with proteinase K digestion (Life Technologies) to extract genomic DNA for genotyping via PCR. We harvested the decapitated larval heads in Trizol (Invitrogen). After genotype confirmation, we pooled 20 heads per biological replicate WT and extracted total RNA. For RNA-seq on human LCLs, we isolated total RNA from transformed lymphoblast derived from cases harboring mutations in FAM50A (p.Trp206Gly and p.Glu254Gly) and unaffected male control. We then treated with a DNA-free™ DNase treatment kit (ThermoFisher Scientific) per manufacturer’s protocol. For RNA quality assurance, we migrated the RNA on screen tape (Agilent Technologies) using a Tape Station 2200 (Agilent Technologies) and the RIN score was determined using Tape Station Analysis Software (Agilent).

Enteroids were collected and Matrigel was dissolved in ice-cold PBS. An RNeasy Plus Micro Kit (Qiagen) was used to extract total RNA from the enteroids, and spectrophotometry (Nanodrop, Thermo Fisher Scientific) was used to determine RNA concentration. A DNA-free DNase Treatment kit (Thermo Fisher) was used to remove gDNA and a First-strand cDNA Synthesis kit (OriGene) was used to synthesize cDNA. A StepOnePlus Real-Time PCR System (Applied Biosystems) with PowerUp SYBR Green Master Mix (Applied Biosystems) was used for PCR amplification. Felis catus-specific primers were designed with Primer Blast (NCBI). Primers were validated with melting curve analysis and amplicon size confirmation via gel electrophoresis. StepOne Software v2 1 was utilized for qPCR analysis with Felis catus GAPDH used as an endogenous control. Primers’ sequences can be found in Supplementary Table S1.