title: NextSeq 500 sequencing; Size variation is regulated differently between and within sexes in natural populations
design: Size variation is regulated differently between and within sexes in natural populations
library_construction_protocol: The DGRP lines used for TWAS were obtained from the Bloomington Drosophila Stock Center. The GD and KK lines for RNAi were obtained from the Vienna Drosophila Resource Center (VDRC), which were to knock down CG7339 (VDRC_ID: 101401), CtsB1 (108315), Hsc70-3 (101766), CG7173 (107756), Dr (110625), CG31075 (101809), CG5590 (109310), CG7519 (110613), Jafrac1 (109514), LM408 (108218), Lsp1alpha (101101), Ppn (108005), Scsalpha (107164), spirit (107936), CG14207 (31800), Top2 (30625), Tpi (25643), yip2 (26562), CG11089 (31420), Dip-B (6296), CG33920 (103447) and CG1315 (47097). hh-Gal4 was obtained from Genetic Strains Research Center, NIG, Japan. Third instar larvae wandering on the wall of the culture vial were dissected in ice-cold Hank's balanced salt solution under the microscope. Wing discs were collected separately for each sex, snap-frozen in liquid nitrogen and kept at -80 °C until use. This process was repeated at different dates and cultures until the total number of wing discs per line/sex/replicate became more than 30 (45 on average). Flies were cultured in a standard condition as previously described (Okada et al., 2016). Wing disc samples in tubes kept at -80 °C were thawed and centrifuged at 6,000 r.p.m. for 2min on table-top centrifuges. The supernatant was removed and the discs in a tube were lysed by pipetting up and down in 100 µl of TRIzol Reagent (ID: 15596018, Life Technologies). The lysate was transferred into the next tube and wing discs were mixed and lysed. This was continued until the last tube, to have more than 30 wing discs lysed. TRIsol was added so that the lysate volume was to be 1 ml. To perform phase separation, 0.2 ml of chloroform was added and vigorously mixed for 15 sec. Following 3 min incubation, the sample was centrifuged at 12,000 × g for 10 min at 4 °C and the upper aqueous phase was transferred into a new tube. To isolate RNA, the sample was mixed with 0.5 ml of 100% isopropanol for 10 min and centrifuged at 12,000 × g for 10 min at 4 °C. After removal of sup, 1ml of 75% ethanol was added slowly and incubated for 15 min at -80°C. Following centrifugation at 7,500 × g for 5 min at 4 °C, the sup was discarded. For thorough cleanup, after being dissolved in 100 µl of RNase-free water, RNA was additionally cleaned using RNeasy mini kit (ID: 74104, Qiagen) following the manufacture's instruction and kept at -80 °C until use. Two RNA-seq libraries were prepared from 128 RNA samples using the Bulk RNA Barcoding and sequencing (BRB-seq) protocol (Alpern et al., 2018) , each of which contained cDNAs made from the 3'-end fragments of transcripts differentially labeled for 64 RNA samples. Briefly, BRB-seq uses the concept of early multiplexing to produce 3' cDNA libraries that allows multiplexing of up to 96 different RNA samples. The protocol is adapted from SCRB-seq, developed for single cell transcription profiling, with several important modifications involving modified oligo-dT for cDNA barcoding and second-strand synthesis with DNA PolII Nick translation. The sequencing library is then prepared using cDNA tagmented by an in-house i7/i7 compatible Tn5 transposase and further enriched by limited-cycle PCR with Illumina compatible adapters.
identifiers.namespace: Laboratory of Systems Biology and Genetics, Ecole Polytechnique Federale de Lausanne (EPFL)  Swiss Institute of Bioinformatics
identifiers.id: E-MTAB-7662:S117_s