GSM9039847: Metaphase stage, biol rep 2; Cryptococcus neoformans; Hi-C
This protocol was adapted and modified from the previously published literature (Rao, Huntley et al. 2014, Belaghzal, Dekker et al. 2017, Golloshi, Sanders et al. 2018). For Hi-C library preparation, approximately 50 OD600 cells from three stages were suspended in 2% YPD and fixed with formaldehyde (3% final concentration) for 20 min and then quenched with 125mM glycine final concentration for another 20 min at room temperature. The fixed cells were washed and resuspended in 1x Tris buffer (50mM Tris-HCl pH 7.5, and 150mM NaCl) with protease inhibitor cocktail, and were lysed using a Bio-spec Bead Beater. The lysate was centrifuged, and the pellet was washed with 1x NEB DpnII buffer. The pellet was digested with DpnII, followed by 14 biotin-dCTP fill in (Jena Bioscience), ligation, decrosslinking and Phenol: Chloroform: Isoamyl alcohol purification, precipitation, followed by RNase treatment as mentioned in(Golloshi, Sanders et al. 2018). DNA was fragmented using a Covaris M220 focused ultrasonicator to produce a size range of 200 to 500 bp. Sheared DNA fragments were fractionated using AMpure XP beads and pulled down using MyOne Streptavidin C1 beads. The on-bead DNA library preparation was carried out using NEBNext Ultra II DNA library preparation kit (E7645) as reported previously (Golloshi, Sanders et al. 2018).