NextSeq 500 paired end sequencing; Poly(A) mRNA-seq from yw and Twi-mCherry-LEXY(V416L) embryos subjected to different blue light exposure schemes
Poly(A) mRNA-seq from yw and Twi-mCherry-LEXY(V416L) embryos subjected to different blue light exposure schemes
yw flies (BL6598) or homozygous Twi-mCherry-LEXY(V416L) flies were grown in medium-sized cages. After three 1 h pre-lays, two consecutive 1 h embryo collections were performed on apple juice agar plates with yeast paste. Afterwards, embryos were subjected to one of two different light exposure schemes. For continuous light exposure, embryos were immediately dechorionated using 50% bleach and spread in a Petri dish containing PBTr (PBS + 0.05 % TritonX-100). Half of the embryos were then kept in the dark, while the other half was exposed to blue light (using a LED illumination box) for 2 h, 3 h, 4 h, or 5 h. For light exposure post-gastrulation, collected embryos were aged for 2 h in the dark before being dechorionated. Stage 8 embryos were then hand-selected under a stereo-microscope, spread in a Petri dish containing PBTr, and kept in the dark or exposed to blue light until 4-5 h and 5-6 h AED (after egg deposition). Finally, for all samples, embryos from the two consecutive collections were combined into a 2-4 h or 4-6 h AED collection, blotted dry, snap-frozen in liquid nitrogen and stored at -80C.   All steps were performed at 25C. Flies and embryos were handled under safelight conditions and only appropriate samples were exposed to blue light at the indicated times. Two or three independent collections were performed for each condition for yw and Twi-mCherry-LEXY(V416L) embryos, respectively.  Fly embryos were homogenised in TRI Reagent (Sigma-Aldrich) using pestles and a cordless motor for pellet mixing and total RNA extraction was performed according to the manufacturer's instructions. Remaining DNA was digested using RNase-free DNase I (Roche), followed by a clean up using the Agencourt RNAClean XP kit (Beckman Coulter) according to the manufacturer's protocol. Poly(A) mRNA isolation and fragmentation were performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer's instruction manual E7420, Chapter 1. 1 ??g total RNA was used as starting material and the protocol optimised for 200 bp RNA inserts was followed.  RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina according to the manufacturer's instruction manual E7420, Chapter 1. For library enrichment, 12 PCR cycles were used. Indexed RNA-seq libraries were pooled in an equimolar ratio and paired-end sequenced with a read length of 75 bp on an Illumina NextSeq 500 High Output Flow Cell.