Name: CAGE_2I_RAL-799r2_68h
Instrument: Illumina HiSeq 2000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: CAGE
Layout: SINGLE
Construction protocol: The 82 genotyped lines were selected from the 205 in the DGRP collection based on three criteria: 1) including lines with the highest quality and depth of genome sequencing, 2) avoiding pairs of highly-related lines and (3) avoiding lines with unusual levels of residual heterozygosity. Freshly enclosed adults were placed in embryo collection vials, with standard apple cap plates. After three/four 1 hr pre-lays, the flies were allowed to lay for 2 hrs, after which the embryos were aged to the appropriate time-point. The embryos were then dochorionated using 50% bleach, snap frozen in liquid Nitrogen and store at -80C. ~100 embryos were homogenized with Cordless Motor for Pellet Mix and pestels (VWR) in ice. RNA was extracted in TRIzol[tm]LS (Life Technologies), digested with RNAse free DNase I (Roche) and purified with the RNeasy mini kit (QIAGEN) according to the manufacturers' recommendations. Production of libraries containing 27bp-tags from 5' ends of capped transcripts. Taken from Takahashi et al, Nat Prot 2012. Briefly, 1-5ug of total RNA are retrotranscribed with a primer containing a common sequence followed by 15 Ns. The RNA-cDNA duplexed is subjected to in-vitro biotinylation of the 5'cap. Then the single-stranded RNA is degraded with RNaseI and the remaining biotinylated (capped) RNA-cDNA duplexes are pull-down with streptavidin-conjugated Dynabeads. cDNA is released through NaOH treatment, and then a barcoded 5'adapter is ligated to the 3'end of the single strand cDNA. Non-ligated adapter is removed by purifying twice with AMPure XP beads. Second strand is synthesized using a primer corresponding to the 5'adapter. Then, 27bp-long tags are generated by digestion with EcoP15I, a type III restriction enzyme. The two matched restriction sites are in the 5'adapter and in the RT primer sequences. A common 3'adapter is then ligated to the resulting fragments. The libraries are amplified by PCR, tipically for 8-15 cycles. After PCR, primers are removed with exonuclease treatment, and the double strand fragments are purified both with QIAquick columns and AMPure XP beads to remove unwanted adapter dimers.