GSM8425892: WT, stigma, α-IgG, replicate 2, Cut&Tag; Oryza sativa; OTHER
About 50,000 cell was harvested and incubated with concanavalin A coated magnetic beads for 15 min at RT. Then, bead-bound cells was resuspended and incubated with the appropriate primary antibody overnight at 4℃. Afterwards, secondary antibody was added to this mixture for 30 min at RT. The magnet stand was used to remove unbound antibodies.pA-Tn5 adapter complex was added at this step at RT for 1 h,then,remove unbound pA-Tn5 protein.Next,cells were resuspended in tagmentation buffer at 37℃ for 1 h. After that, immunoprecipitated DNA was saved. To amplify libraries, 21 µL DNA was mixed with 2 µL of a universal i5 and a uniquely barcoded i7 primer, using a different barcode for each sample. A volume of 25 µL NEBNext HiFi 2× PCR Master mix was added and mixed. The sample was placed in a Thermocycler with a heated lid using the following cycling conditions: 72 °C for 5 min (gap filling); 98 °C for 30 s; 14 cycles of 98 °C for 10 s and 63 °C for 30 s; final extension at 72 °C for 1 min and hold at 8 °C. Library was sequenced on Illumina novaseq 6000 with PE 150 method.