Anaesthetized adults were placed in a 15ml conical tube, flash frozen in liquid nitrogen, then shaken vigorously for 10 seconds. Broken flies were placed on a Petri dish on dry ice and frozen severed heads were removed with forceps to a fresh tube. Flies were processed in groups of 100 animals per dissection. Typically, heads were missing the antennal and maxillary organs, while the mouth-parts were retained.
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):83
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).
FlyBase reports gene expression levels calculated from RNA-Seq coverage data as RPKM (reads per kilobase of exon model per million mapped reads). The RPKM value is calculated as follows. The uniquely transcribed region(s) for each gene is determined by taking regions covered by exons of the gene and excluding transcribed regions from any overlapping genes, both with respect to genes lying on same strand (for calculation using strand-specific RNA-Seq coverage data), and for genes on either strand (for calculation using unstranded RNA-Seq coverage data). RNA-Seq coverage read-count data was then correlated by location with the uniquely transcribed region(s) of each gene to produce the sum of reads over the entire uniquely transcribed region for the gene. Reads per kilobase of exon model per million mapped reads (RPKM) was then calculated using the method from Motazavi et al, Nat. Methods 6, 621-628 (2008). (RPKM = 10^9 * C / N * L * R, where C = number of reads in gene, N = number of uniquely mappable reads in the experiment, L = sum of uniquely transcribed bases in bp, and R = read length in bp).
The RPKM values are binned into eight expression levels: Bin 0: No/Extremely low expression (0); Bin 1: Very low expression (1-3), percentiles 1-25, approximately; Bin 2: Low expression (4-10), percentiles 26-50, approximately; Bin 3: Moderate expression (11-25), percentiles 51-75, approximately; Bin 4: Moderately high expression (26-50), percentiles 76-85, approximately; Bin 5: High expression (51-100), percentiles 86-95, approximately; Bin 6: Very high expression (101-1000), percentiles 96-99, approximately; Bin 7: Extremely high (>1000), the 100th percentile, approximately.
FlyBase RPKM data for all genes can be downloaded from the FlyBase Downloads page (link in the blue navigation bar at the top of all FlyBase web pages).
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.
