Nnk null 24h AEL, rep3
Whole larvae (~30 animals at 24 hrs AEL and ~20 animals at 48 hrs AEL for each sample; RNA from each time point and genotype was prepared in triplicate) were ground in a 1.5mL Eppendorf tube containing 50uL of TRIzol reagent using a DNase, RNase and DNA free 1.5mL pestle. 450uL of TRIzol reagent was added after grinding. Immediately, we added 500uL of chloroform and the tube was inverted gently 2-3 times. We removed the aqueous phase into a fresh tube containing 1mL of 200 proof EtOH and mixed by inversion. The mixture was then bound to a Zymo-spin column according to the manufacturer's instructions (Zymo Research). We followed the DNase extraction and purification protocol outlined in the RNA Clean and Concentrator kit (Zymo Research). We eluted the RNA in 15uL of DNase/RNase-free water and immediately placed the samples at -80C. We checked the quality of the samples with a 2200 Tapestation (Agilent Technologies) and selected samples that had an RNA integrity number > 9.0 for library preparation. Library construction and Illumina 150-bp paired-end RNA-sequencing were conducted at Novogene Bioinformatics Technology Co., Ltd (Beijing, China).