HiSeq X Ten paired end sequencing; Chromatin Accessibility Dynamics During Zebrafish Early Embryogenesis At The Single-Cell Resolution
Chromatin Accessibility Dynamics During Zebrafish Early Embryogenesis At The Single-Cell Resolution
Staged embryos were transferred into 10cm dish on ice. The chorion was physically disrupted and removed by tweezers. Then the majority of the yolk was physically removed using a tweezer. The remaining embryonic cells were washed with PBS and incubated in the deyolk buffer. When single cells became dissociated, they were transferred into a new eppendorf tube and washed with PBS for three times. Nuclei acid was extracted first using a Qiagen PCR purification column. Then 1× VAHTS DNA cleaning beads were used to remove primer leftover and eluted in 20 μl Nuclease-free Water. 50,000 single cells were resuspended in 50 uL RSB + 0.01% digitonin 0.1% Tween-20 and 0.1% NP-40 and incubated on ice for 3 minutes. Then 1 mL of cold RSB + 0.1% Tween-20 (without digitonin and NP-40) was added. The permeabilised cells were centrifuged at 300 g 4 C. After removing the supernatant, 50 ul tagmentation mix was used to resuspend the cell pellet and incubated at 37C on a thermomixer for 30 minutes. Then 50 ul 20mM EDTA was added to stop the reaction. Single cells were sorted into each well of a plate containing distinct well index primers using FACS. 2x NEBNext PCR Master Mix was added into each well to perform PCR for the library construction. The PCR condition is: 72 C 5 minutes, 98 C 2 minutes, then 12 cycles of [98 C 10 seconds, 63 C 30 seconds, 72 C 20 seconds].