Protocols: 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. The 3' TagSeq was performed with the following modifications: 3ug of total RNA were fragmented by incubation at 94C for 5 min in RNA fragmentation buffer (200 mM Tris acetate pH 8.2, 500 mM potassium acetate and 150 mM magnesium acetate). The reaction was stopped with 0.045M ethylenediaminetetraacetic acid (EDTA) pH 8 (final concentration). The fragmented RNA was precipitated in ethanol and resuspended in 8ul nuclease free H2O. For retrotranscription, the 8ul of fragmented RNA were mixed with 1ul of biotinylated oligo P7_dT16VN (50 mM) (BioSpring) and 1 ul of 10 mM dNTPs. The samples were incubated at 65C for 5 min and transferred to ice for 1 min. Four microliters of 5X first-strand buffer (Life Technologies), 2 ul DTT 0.1 M (Life Technologies), 0.1 ul actinomycin D (1.25 mg/ml), 1ul RNaseOUT Recombinant Ribonuclease Inhibitor (Life Technologies), 2ul nuclease free H2O and 1 ul Superscript III were added to each sample, and incubated at 50C for 50 min. The samples were immediately purified using 1X of Ampure XP beads and eluted in 40 ul elution buffer (EB)(10 mM Tris-HCl, pH 8). Second-strand cDNA synthesis was performed according to the manufactures suggestions (Fermentas) and incubated at 15C for 3h. Fragments from 200-500 bps were size selected with Ampure XP beads. Binding to streptavidin beads (Dynabeads M-280 Streptavidin, Life Technologies) was performed according to the manufactures protocol . End repair, (A) tailing and ligation of Illumina sequence adaptor P5- P7_T1_Mpx (1ul of 1.25uM) were performed with the NEBNext DNA Library Prep Reagent Set for Illumina (NEB) according to the manufacturers recommendations. Beads were washed every step according to the protocol: twice with 200ul 1X B&W buffer (5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA and 1M NaCl), once with 200 ul EB. Samples were resuspended in 30ul EB. Enrichment polymerase chain reaction (PCR) was performed using 15 ul of beads, 10ul 5X Phusion HF Buffer (Thermo Scientific), 1 ul 10 mM dNTPs, 1 ul each of oligos PE1.0 and PE2.0 (10mM; Illumina), 1 ul Phusion DNA Polymerase (Thermo Scientific) and 22 ul H2O. The PCR program was 30s at 96C, 16 cycles of (10s at 96C, 10s at 60C and 30s at 72C) and 5 min at 72C. Fragments from 200-500 bps (average size 250-270 bps) were size selected with Ampure XP beads and eluted in 17 ul of EB. Library quality was assessed on a 2100 Bioanalyzer system (Agilent).