Comparison of ATAC seq between control wing imaginal discs and salm/salr knockdown wing imaginal discs
Comparison of ATAC seq between control wing imaginal discs and salm/salr knockdown wing imaginal discs
We used 20 imaginal wing discs per sample to make two technical replicates, and the larvae were of salEPV-Gal4 UAS-salm-i/+; UAS-salr-i/+ and salEPV-Gal4 UAS-GFP/+ genotypes. The discs were dissected in pre-cooled PBS, pipetted in PBT (PBS, 0.1 % Triton X-100) containing 1x proteases inhibitors (cOmpleteâ„¢ Protease Inhibitor Cocktail tablets, Roche, 11873580001) and suspended in transposition solution containing. Disk were permeabilization: Thaw disks on ice for 10 min and add 50ul PBT+PI; Add 50 ul of PBT + 0.4% IGEPAL CA-630; Incubate at 4C for 30 min on a nutator and Spin at 3200 xg for 5 min at 4C. Tagmentation and reverse-crosslinking: Resuspend the nuclei pellet in 50 ul tagmentation mix(5 uL TDE1 (Illumina, 15027865), 25 uL 2x buffer (Illumina, 15027866), 20 uL nuclease-free H2O). Incubate at 37C for 30 min. Add 50 ul STOP buffer, 5 ul of 1mg/ml RNase A and incubate at 55C for 10 min. Add 3 ul Proteinase K (20 mg/ml) incubate 65C for 1 hour. Purify with Zymo DNA clean and concentrator kit. Finally samples are PCR amplified and size selected with SPRI beads.