Name: GSM7476867
Instrument: NextSeq 500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: High molecular weight (HMW) genomic DNA was used as input for all Nanopore and bulk Illumina sequencing experiments. For extraction, bulked seedlings were dark treated for 1 week prior to tissue collection. Four grams of frozen tissue was ground under liquid N2 and pre-washed twice with 1.0M sorbital. The tissue was then transferred to 20ml pre-warmed lysis buffer (100mM Tris-HCl pH 9.0, 2% w/v CTAB, 1.4M NaCl, 20mM EDTA, 2% PVP-10, 1% 2-mercaptoethanol, 0.1% sarkosyl, 100ug/mL proteinase K), mixed gently, and incubated for 1 hour at 65˚C. Organic extraction in phase-lock tubes was performed using 1 vol phenol:chloroform:isoamyl alcohol (25:24:1) followed by 1 vol chloroform:isoamyl alcohol. DNA was precipitated by adding 0.1 vol 3M NaOAc pH 5.2 followed by 0.7 vol isopropanol. HMW DNA was hooked out with a pasteur pipette and washed with 70% EtOH, air dried for 2 minutes, and resuspended in 200ul Tris-Cl pH 8.5 (EB). The solution was treated with 2ul 20mg/ml RNase A at 37˚C for 20 minutes followed by 2ul 50mg/ml proteinase K at 50˚C for 30 minutes. 194ul EB, 100ul NaCl, and 2ul 0.5M EDTA were added and organic extractions were performed as before. DNA was precipitated with 1.7 vol EtOH, hooked out of solution with a pasteur pipette, washed with 70% EtOH, and resuspended in 50ul EB. HMW DNA from separately maintained Tpd1;Tpd2 lineages (BC5S3, BC8S3) and bulk segregation analysis (BSA) pools were extracted as detailed above. Libraries were prepared using the Illumina TruSeq DNA PCR-Free kit (Illumina, cat#20015962) with 2ug of DNA input.