GSM8881990: Zea mays gDNA homozygous N siblings backcrossed into W22; Zea mays; OTHER
Leaf discs were collected from 80 Trf and N siblings in the W22 backcross population (at least 10 introgressions). High-quality gDNA was extracted using a urea-based extraction method which is as follows. Tissue was ground in urea extraction buffer (4M urea, 4M NaCl, 1M Tris pH8, 0.5M EDTA, 34mM n-lauroyl sarcosine). Then an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), was added and vortexed to mix. After centrifugation, the supernatant was decanted and mixed with an equal volume of chloroform. After centrifugation, the supernatant was decanted and mixed with 1/10th volume of 4.4 M NH4OAc pH 5.2 and 0.7 volume of isopropanol. Strands of DNA were then collected and washed with 70% ethanol in a separate tube. The dried pellet was resuspended in 100μl of TE. DNA integrity was checked using gel electrophoresis and quantified using qubit before sending for library preparation and 150bp PE sequencing by Novogene. Sequencing was for 10 x coverage of the maize genome. A total amount of 1.0μg DNA per sample was used as input material for the DNA sample preparations. Sequencing libraries were generated using NEBNext® DNA Library Prep Kit following manufacturer's recommendations and indices were added to each sample. The genomic DNA is randomly fragmented to a size of 350bp by shearing, then DNA fragments were end polished, A-tailed, and ligated with the NEBNext adapter for Illumina sequencing, and further PCR enriched by P5 and indexed P7 oligos. The PCR products were purified (AMPure XP system) and resulted libraries were analyzed for size distribution by Agilent 2100 Bioanalyzer and quantified using real-time PCR.