White prepupae were collected and dissected in saline by tearing them immediately posterior to the mouth hooks using paired forceps and everting them. Males and females (as identified by the clear gonadal tissue embedded in fat body of the A5 segment) were dissected separately, and preparations were combined in equal numbers for RNA processing. Salivary glands were isolated, removing as much fat body as possible, and frozen on dry ice.
Total RNA was isolated using Trizol, then Qiagen RNeasy spin columns. mRNA was isolated using Dynal oligo(dT) beads, then fragmented using divalent cations under elevated temperature, followed by sequential ligation of RNA linkers to the 5’ and 3’ ends. Next, reverse transcription was performed using a primer complementary to the 3’ linker and PCR was performed using primers complementary to both linkers. ~ 300 bp fragments were isolated from an agarose gel and gel-purified again.
Read length (bases):79
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).
Reads were aligned to Dmel_Release_6 using the STAR aligner v2.3.0e (Linux x86_64) with default parameters on the FASTQ files to generate multiply-mapped BAM files. These were filtered to include reads with only 1 aligned hit ( NH:i:1 attribute) to generate uniquely-mapped BAM files. A custom script was used to convert BAM files into bedgraph files (bam2bedgraph.cc).
The RNA-seq profiles displayed by FlyBase in GBrowse and used for RPKM calculation can be accessed at the FTP link below as .wig files. Please take note of how these FlyBase .wig files represent data for a contiguous sequence of bases with the same signal value. The value is declared only for the first position of that region, and applies to all positions that follow (these are not explicitly listed) until a new value at a new base position is declared.