After incubation for 10 minutes at room temperature, the sample was transferred to a magnetic separator (DynaMag-2 magnet Life Technologies), and the supernatant was transferred to a new Eppendorf tube. A double size selection using AMPure XP beads (Beckman Coulter) was performed: first, the ratio of AMPure XP beads solution volume to DNA sample volume was adjusted to 0.6:1. dATP was added with Klenow exo- (NEB) for 30 minutes at 37˚C, and the enzyme was heat-inactivated (20 minutes at 65˚C). The sonicated DNA was end-repaired (T4 DNA polymerase, T4 DNA polynucleotide kinase, Klenow (all NEB)) for 30 minutes at 20˚C, followed by DNA purification (Qiagen PCR purification kit). Sonication was performed to obtain DNA fragments with a size peak around 400 bp (Covaris E220 settings: duty factor: 10% peak incident power: 140W cycles per burst: 200 time: 110 seconds). DNA was eluted in 130 μl double distilled water. Each 5 µg of Hi-C library DNA were incubated with 0.1 mg/ml BSA, 1x NEBuffer 2 (NEB), 0.2 mM dATP, and 15 units T4 DNA polymerase (NEB) in a total volume of 100 μl for 4 hours at 20˚C to remove any biotin from non-ligated fragment ends, followed by DNA purification (Qiagen PCR purification kit). After three consecutive washes with 70% v/v ethanol, the DNA concentration was determined using Quant-iT Pico Green (Life Technologies). The DNA was precipitated in the presence of sodium acetate 3 M pH 5.2 (1/10 volume) and absolute ethanol (2.5 x volumes) overnight at -20˚C. The DNA extraction was repeated with one phenol extraction followed by two sequential phenol/chloroform extractions then one chloroform extraction. The DNA was precipitated in the presence of sodium acetate 3 M pH 5.2 (1/10 volume) and ethanol (2.5 x volumes) 45 min at -20˚C. Reverse crosslinking (65˚C overnight in the presence of Proteinase K (Roche)) was followed by RNase A (Roche) treatment at 37 ˚C for 60 minutes and two sequential phenol/chloroform extractions. Filled-in chromatin was mixed with 0.93 volumes of ligation reaction mix at a final concentration of 1x ligation buffer (NEB), 0.1mg/ml bovine serum albumin (BSA), 25 units / sample T4 DNA ligase (Life Technologies) for 6 hours at 16˚C. The restriction sites were then filled in with Klenow (NEB) using biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (all at a final concentration of 0.28 µM) for 60 minutes at 37˚C. Restriction digest was performed overnight at 37˚C with HindIII (NEB 1500 units per sample). Triton X-100 was added at a final concentration of 0.95 % and incubated at 37˚C with shaking (950 rpm) for 10 minutes. Lysed cells were centrifuged at 4000 g at 4☌ for 10 minutes and resuspended in 0.38 volume of the adjusted OD600=10.0 with 1x NEBuffer 2, SDS was added (0.1% final concentration) and the chromatin of mechanically lysed cells were incubated at 65˚C with shaking (950 rpm) for one hour. The OD600 of the pellet was adjusted to 10.0 with 1x NEBuffer 2 followed by 2 volumes washes with 1x NEBuffer 2. Lysed cells were washed in 50 ml 1x NEBuffer 2 then centrifuged at 3000 rpm at 4☌ for 5 minutes. The pellet was re-suspended in 5 ml 1x NEBuffer 2.Ĭells were ground by mortar and pestle in liquid nitrogen for 10 minutes. Cells were centrifuged at 3000 rpm at 4☌ for 5 minutes, washed in 100 ml sterile water, then re-centrifuged at 3000 rpm, 4☌ for 5 minutes. Fixation was done with formaldehyde at 3% with shaking at 220 rpm, 37☌, then quenched with glycine at 0.35M final concentration and 5 min incubation at 37☌. of fresh YPD, prewarmed to 49˚C, and grown with shaking at 37˚C for 15 minutes. The other group was shifted to heat-shock conditions by adding 1 vol. The reaction was quenched with 2.5M glycine added to a final concentration of 0.35M and incubated for 5 minutes with shaking at 220 rpm, 25☌. One fixed with formaldehyde (Agar Scientific) at a final concentration of 3% for 20 minutes with shaking at 220 rpm at 25☌. The culture was diluted down to OD600=0.2 in 400 ml total culture volume and incubated at 25☌ with shaking at 200 rpm until mid-log phase (OD600=0.4-0.6). The starter culture was diluted back down to OD600=0.2 in a 100 ml intermediate culture and incubated o/n at 25☌ with shaking at 200 rpm. A single colony was selected and grown overnight in 6 ml yeast peptone dextrose (YPD) medium at 25☌ with shaking at 200 rpm in an incubator (Infors HT Multitron Pro). The plates were grown in an incubator (Heraeus) at 25☌ for 3 consecutive days. Cells were inoculated from a frozen glycerol stock onto appropriate medium plates using inoculation loops. Saccharomyces cerevisiae BY4742 was used for all experiments. GEO help: Mouse over screen elements for information.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |