title: Illumina NovaSeq 6000 paired-end Sequencing of KAS22026661 (KAE22281120)
design: missing
library_construction_protocol: Library preparation process was conducted based on the previous reported method Corces et al., 2017. 75,000 cells were harvested and treated with 50 ul of cold RSB 10 mM pH 7.4 Tris-Cl, 10 mM NaCl, 3mM MgCl2 containing 0.1% NP40, 0.1% Tween-20 and 0.01% Digitonin for 3 minutes on ice. Cells were washed with cold RSB containing 0.1% Tween-20 and pelleted to aspirate supernatant. Cell pellet was resuspended in 50 ul transposition mixture 25 ul 2x TD buffer, 2.5 ul transposase, 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul DW 2x TD buffer 20 mM pH 7.6 Tris-Cl, 10 mM MgCl2, 20% dimethyl formamide. Transposition reaction was done at 37 degree celsius for 30 minutes with consistent mixing. The reaction was terminated by adding 50 ul tagmentation stop buffer 10 mM pH 8.0 Tris-Cl, 20 mM pH 8.0 EDTA. The products were purified in 21 ul of elution buffer using Zymo DNA clean and Concentrator-5 Kits. Eluted DNA was amplified for five cycles using NEBnext high-fidelity 2X PCR master mix. Indexing primers were from Nextera DNA library prep kit. Using 5 ul of the pre-amplified mixture, 15 ul qPCR was performed to calculate the additional number of PCR cycles needed for library preparation. Linear cycle and relative fluorescence were plotted and the cycle number that corresponds to quater of maximum fluorescent intensity was determined. The remaining pre-amplified mixture was amplified with qPCR with the determined additional cycles. The final reactants were purified using Zymo DNA clean and Concentrator-5 Kits. Sequencing was performed with NovaSeq 6000 platform by 101 bp paired-end.