title: genomic sequencing for line NY66-2
design: The genomic DNA (gDNAs) used for the library preparations were extracted with the Gentra Puregene Cell Kit including the RNase  A Solution (Qiagen) or the reagents from the Puregene Accessories (Qiagen, as indicated below). The extraction and purification procedures were modified from the protocol for Drosophila Melanogaster (Qiagen Supplementary Protocol) and combined with column purification (Zymo) as described below, to give better and more constant yields with higher throughput.     About 15 frozen (-80\textcelsius) female flies of each fly line were homogenized into small pieces in cold 300$\mu$L Cell Lysis Solution (158906, Qiagen), and then incubated for 15min at room temperature. The RNAs were digested by 4.5ul RNase A Solution (158922, Qiagen) and incubated for 15min at 37\textcelsius {}. The contaminated proteins were precipitated by mixing with 100$\mu$L Protein Precipitation Solution (158910, Qiagen) and centrifuged 4min at 16,000g. The extracted gDNAs in the supernatant were column purified with the DNA Clean and Concentrator –25 Kit (D4013, Zymo) followed its protocol, using 5 volumes of Binding Buffer. The quality of these purified gDNAs were checked with 1\% agarose gel, showed intact size about 10kb without degraded lower bands.     About 3$\mu$g purified gDNAs in 100$\mu$L EB (Elution Buffer, 10mM Tris, pH8.5, Invitrogen) were sheared with Covaris at 300bp setting and purified with Purelink PCR Purification (HC) Kit (K3100-01, Invitrogen), followed their protocols respectively. The distributions of these fragments about 300-1000bp were confirmed with 2\% agarose gel.    The purified fragments were end-repaired for 30min at 25\textcelsius {} with Quick Blunting Kit (E0542L, NEB), A-tailed for 30min at 37\textcelsius {} with Klenow Fragments (M02112L, NEB) and dATP (R0141, Fermentas), ligated to adaptors (see below) for 10min at 25\textcelsius {} with Quick Ligation Kit (E0542L, NEB), size selected between 350bp to 450bp on 2\% agarose gel Seakem LE (5000, Lonza) and extracted with ZymoClean Gel DNA Recover Kit (D4001, Zymo), and PCR amplified 15 cycles by Phusion High-Fidelity DNA Polymerase (M0530S, NEB) with dNTP (N0447L, NEB) and Primers (see below), followed these reagent’s protocols respectively. The DNA Clean and Concentrator-5 Kits (D4013, Zymo) were used to purify the product after each reaction.    The libraries showed a band between 400bp to 500bp on 2\% agarose gel. All concentrations through the preparation were measured with Qubit Fluorometer (Invitrogen). Final libraries were diluted to 10nM before sequencing, based on the reads from Qubit and the sizes from agarose gel.    Adapters and PCR Primers for Paired-End Library:  All adapters and primers were HPLC purified and ordered from Integrated DNA Technologies (IDT):\\      \footnotesize \noindent PE-Ad1, 5'-/5Phos/GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG \\  PE-Ad2, 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T \\  PE-PCR, (1)  5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T \\  PE-PCR, (2)  5'-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC*T \\