sciATAC_SS157
Protocols: The virginizer flies of 5-10 days age were transferred into fresh bottles and left to lay embryos for about 24 hours at 20C before the adults were removed from the bottles. The bottles were heat-shocked inside a 38C water bath for 1 hour. The virgins eclosed from heat-shocked bottles of the virginizer line were collected, mated to the hand-picked males from the paternal lines by putting all flies into cylindrical fly houses in a 25C room. The flies were left in the cages for 2 days before starting the collections. On the day of collection, one hour prelays were done for 3 times, followed by 2-hour collections. The plates with embryos were aged to the stages desired at 25C. The embryos were washed onto the set of sieves and dechorionated by incubating in the 50% bleach for 2.5 min. Around 200 embryos were transferred into an eppendorf tube and snap-frozen in liquid nitrogen for RNA assays. The majority of embryos were fixed by incubating in 9.5 ml cross-linking solution with 485 microliters 37% formadehyde with vigorous shaking for 15 min. Cross-linking was stopped by incubating with Glycine solution with vigorous shaking for at least 1 min. After washing with PBT, the embryos were blotted dry. The cross-linked dry embryos were transferred into 1.5 mL eppendorf tubes, snap-frozen in liquid nitrogen and kept in -80C for ATAC-seq and iChiP. Frozen embryos were transferred into a glass douncer placed on ice and thawed in HB buffer with proteinase inhibitor. Embryos were dounced 20 times with the loose pestle and 10 times with the tight pestle. The homogenate was filtered through two layers of Miracloth, centrifuged and the supernatant was discarded. The nuclei pellet was washed once in HB buffer and resuspended in PBT. The nuclei were dissociated by passing them 10 times through a 20G needle and 10 times through a 22G needle using a 5 mL syringe and then filtered through 20 µuem Nitex membrane. The extracted nuclei were quantified using the LSRFortessa system, snap frozen in liquid nitrogen and stored at -80C. sciATAC-seq was performed largely as described in (Cusanovich, Reddington, Garfield et al. Nature. 2018). In summary, frozen nuclei were thawed, washed in PBT and dispensed in a 96 well plate by FACS (2500 nuclei / well). Tagmentation was performed by addition of Tn5 loaded with unique barcode combinations per each well and by incubation at 55 C for 1 hour. The tagmentation was stopped by addition of EDTA and incubation at 37 C for 15 min. Nuclei were pulled and dispesed in 96-well plates by FACS (25 nuclei / well). After reverse-crosslinking at 65 C for 16 hours, PCR amplification of tagmented DNA was performed by addition of uniquely indexed primers, KAPA HiFi DNA Polymerase ReadyMix (Roche) and Bst3.0 (NEB) and with the following cycling conditions: 72 °C 5 min, 98 °C 30 s; 98 °C 10 s, 63 C 30 s, 19–22 cycles; 72 °C 1 min, hold at 10 °C. The optimal number of cycles for each library was determined beforehand by monitoring amplification on a qPCR machine for a set of test wells.