GSM1159630: am30; Drosophila melanogaster; RNA-Seq
We identified GAL4 lines with regional expression, then dissected and sectioned gut regions based on the presence or absence of GFP under a fluorescent dissecting microscope.   Gut regions were isolated, no more than 5 at a time, and moved to iced tripure (Roche/Boehringer Mannheim cat: 11667157001) to avoid RNA degradation (which occurred unless guts were processed within 30 min of isolation (or 10-15 minutes in the case of the copper region samples).  After 25 or 50 gut regions were collected in 200μl tripure, they were homogenized.  600μl of fresh tripure was added, mixed, and allowed to stand at room temperature for 5-10 min.  After adding 180μl chloroform, samples were vortexed 2 x 45 secs, and incubated at room temperature for 10 minutes.  Samples were then centrifuged for 15 min at 12,000 rpm at 4°C, and the aqueous layer was moved to a fresh tube.  RNA was precipitated by adding 400μl of isopropanol, vortexing for 15 seconds, and incubating at room temperature for 15 min, and centrifuging at 12,000 rpm for 15 min at 4°C.  After washing the pellet with 75% EtOH, the RNA was dried in air dry for 5 minutes, resuspended in 50μl nuclease free H2O, and stored at -80°C. cDNA libraries were constructed from poly(A)-selected RNA using Illumina TruSeq RNA Library Prep Kit v2
GEO Accession
GSM1159630