ChIPseq of C. neoformans KN99deltaH1 (H3K4me2)
Freeze-dried cell pellets were broken into fine powder with 0.5 mm zirconia/silica beads in a FastPrep 24 homogenizer (MP Biomedicals) without buffer for three cycles (50s at 8m/s followed by 90s chilling on ice) and then with 250 l of ChIP lysis buffer for another four cycles. The lysate was recovered on ice. Lysate was then diluted to 1 ml with ChIP lysis buffer without detergents and transferred to a milliTUBE 1 ml with AFA Fiber. Samples were sheared with the Covaris E220 Focused ultrasonicator for 13 minutes at 5C. The sheared chromatin was centrifuged at 21000 g for 15 min at 4C to remove insoluble cellular debris. Detergents concentration of the chromatin suspensions was adjusted to 1% Triton X-100 and 0.1% Deoxycholate in order to perform immunoprecipitation in ChIP lysis buffer. Immunoprecipitation was carried out with protein A/G agarose beads washed twice in ChIP lysis buffer without protease inhibitors. The sheared chromatin was subjected to pre-clearing to reduce proteins that non-specifically bind beads by incubation with 30 l of washed agarose beads for 1 hour at 4C on a rotator. After centrifugation at 5000 rpm for 1 min at 4 to pellet the beads, the supernatant was transferred to a new tube and incubated overnight with 20 l of washed beads and either 3 l of H3K4me2 antibody (Active Motif, 39141) or 2 l of H3K9me2 antibody (Abcam, ab1220) at 4C on a rotator. The next day, beads were washed twice in 1 ml ChIP lysis buffer without protease inhibitors, once in 1 ml ChIP lysis buffer containing 0.5 M NaCl, once in 1 ml LiCl wash buffer, and twice in 1 ml TE buffer, all at 4C. The bound chromatin was eluted in 62.5 l of ChIP TES buffer at 65C for 10 minutes. After centrifugation, supernatant was transferred to a new tube. A second elution with 62.5 l of ChIP TES buffer was performed so that the final elution volume was 125 l. 105 l ChIP TES buffer was added to each Input sample. All Input and IP samples were incubated for 16 hours at 65C for reverse crosslinking. After reverse crosslinking, samples were treated with 1,25 l of RNase A (20 mg/mL) and incubated for 2 hours at 37C in a thermomixer at 800 rpm, followed by treatment with 6.25 l of Proteinase K (20 mg/mL) for 2 hours at 42C at 800 rpm. DNA was recovered using a Qiagen PCR purification kit, buffer QG was used to bind DNA and the washing steps were done three times using 500 l of PE buffer. The DNA was eluted in 49 l buffer EB for Input DNA samples and 47 l buffer EB for ChIP DNA samples.