Fly stocks were reared in bottles at 24oC were supplemented with 0.05% bromophenol blue to mark the gut contents of feeding animals. Wandering larvae with clear (emptied) guts, corresponding to puff stage 7-9, were collected and frozen on dry ice.
Frozen samples were homogenized and extracted using the TRIzol reagent protocol (Invitrogen). RNA was purified on an RNeasy spin column (Qiagen), and DNase treated. Polyadenylated RNAs were purified from total RNA extracts via oligo(dT) binding, using standard Illumina protocol. The poly(A)+ RNA was fragmented using divalent cations under elevated temperature, following by first and second strand cDNA synthesis primed with random hexamers. The cDNA fragments were end-repaired using T4 DNA polymerase and Klenow DNA polymerase, and phosphorylated at their 5' ends with T4 polynucleotide kinase. After adding A bases to the 3' end of the DNA fragments, Illumina adaptor oligonucleotides were ligated to the ends and ~ 300 bp fragments were isolated from an agarose gel, enriched by PCR amplification, and gel-purified again.
Read length (bases):76
The samples were quantitated using a Nanodrop, and loaded onto a flow cell for cluster generation and sequenced on an Illumina Genome Analyzer II using either single read or paired end protocols (Illumina).