GSM7817907: leaf, cenh3-4/WT, JZ336; Glycine max; OTHER
For library preparation of genome-resequencing, genomic DNA was prepared using a modified CTAB protocol. 1cm2 leaf tissue was stored at −80ºC before use. The tissue was ground in centrifuge tube cooled by liquid nitrogen using 0.5mm diameter small steel ball and tissue grinder at 30Hz for 15sec twice. The CTAB buffer was preheated to 65ºC in a water bath and 600μL hot CTAB buffer was added to dissolve tissue powder. After incubating at 65ºC for 30min, the liquid was short centrifuged and 500μL supernatant was moved to a clean centrifuge tube. 500μL chloroform: isoamyl alcohol (24:1) was added and mixed gently for 5min. The mixture was centrifuged for 20 min at 12000rpm and 400μL supernatant was taken out to mix with 270μL isopropanol and 30μL 3M sodium acetate (pH=5.6) in a clean centrifuge tube. After -20°C overnight, the liquid was centrifuged for 50 min at 12000rpm and the sediment was washed once using 800μL 70% ethanol. The dried sediment was dissolved with 100μL ddH2O and 1μL RNase was added to degrade RNA at 37 ºC for 1h. The solution was stored at −80ºC for library preparation. 2 μg of DNA were used for a PCR-free library preparation with a PCR-free protocol BGI-NGS-JK-DNA-003. Samples were subjected to quality control according to sample type and products requirements. Genomic DNA was randomly fragmented. The fragmented genomic DNA was selected to a certain average size. The fragments were subjected to end-repair and then was 3' adenylated. And then adaptors were ligated to the ends of these 3' adenylated fragments. The corresponding library quality control protocol will be selected depending upon product requirements. Single-stranded adaptor ligation products are produced via denaturation. The reaction system and program for circularization are subsequently configured and set up. Single-stranded cyclized products are produced, while uncyclized linear DNA molecules are digested. Single-stranded circle DNA molecules are replicated via rolling cycle amplification, and a DNA nanoball (DNB) which contain multiple copies of DNA is generated. Sufficient quality DNBs are then loaded into patterned nanoarrays using high-intensity DNA nanochip technique and sequenced through combinatorial Probe-Anchor Synthesis (cPAS).