Pacbio SEQ2 Short insert library: Drosophila melanogaster Adults
Fly stocks were maintained under standard culture conditions. The w1118 strain was used to collect adults. Genomic DNA was extracted as described previously (Yao et al.,2018). Briefly, to isolate the gDNA, lysis buffer containing 100mM Tris-HCl (pH 8.0), 5mM EDTA (pH 8.0), 0.2% SDS, 200mM NaCl, and 20-50 uL of protease K (20mg/ml) was added to the fly samples, homogenized and incubated at 55 C overnight. After the overnight digestion, the lysates were brought to room temperature, and incubated with 5-10 uL of RNase A solution (20mg/ml) for at least 1 hour at 37 C. DNA was extracted by adding equal volume of phenol: chloroform: isoamyl alcohol at a ratio of 25:24:1 and centrifuging at 12,000 rpm for 5 min. Supernatant was transferred to clean tubes and mixed with 10% of 3M NaOAC and two times of 100% Ethanol at -80 C for at least 30 min. The gDNA pellets were collected by 12,000 rpm centrifugation for 15 min. After washing the gDNA pellet with 70% ethanol, the pellets were dissolved in 30-50 uL of nuclease-free water. The short-insert DNA SMRTbell library was performed according to the manufacturer's instructions by first shearing the sample to ~200bp using a Covaris LE220 Focused-ultrasonicator, followed by damage and end repair and ligation of single-stranded barcoded adaptors. Sequencing primer was then annealed to the SMRTbells and incubated with the template for 1 hour at 20 C using a ratio of 20:1. The Sequel 2.1 polymerase was then bound to the annealed template complex using a ratio of 30:1 polymerase to SMRTbell at 0.5 nM for 4 h at 30 C. The binding complex was cleaned up, diluted to the final loading concentration, and held at 4 C until diffusion-based loading on the Sequel II instrument. Diluted binding complex at concentration of 125-175pM was loaded on the 8M sequencing chip and 30 hour movies were recorded according to the manufacturer's guidelines. The short-insert DNA SMRTbell library was performed according to the manufacturers instructions by first shearing the sample to ~200bp using a Covaris LE220 Focused-ultrasonicator,followed by damage and end repair and ligation of single-stranded barcoded adaptors. Sequencing primer was then annealed to the SMRTbells and incubated with the template for 1 hour at 20 C using a ratio of 20:1. The Sequel 2.1 polymerase was then bound to the annealed template complex using a ratio of 30:1 polymerase to SMRTbell at 0.5 nM for 4 h at 30 C. The binding complex was cleaned up, diluted to the final loading concentration, and held at 4 C until diffusion-based loading on the Sequel II instrument. Diluted binding complex at concentration of 125-175pM was loaded on the 8M sequencing chip and 30 hour movies were recorded according to the manufacturers guidelines.