GSM4595533: ChIP HP1 Input repl2 cycle 9-13 with spike in; Drosophila melanogaster; ChIP-Seq
To extract the nuclei the embryos were transferred into Lysis buffer (140 mM NaCl, 15 mM HEPES [pH 7.6], 1 mM EDTA, 0.5 mM EGTA, 1 % TritonX100, 0.5 mM DTT, 0.1 % Sodium Deoxycholate, 10 mM Sodium Butyrate, 0.1% SDS, 0.5% N-Laurosylsarcosine,1X  Protease Inhibitors) and subjected to ultrasound treatment (Covaris E220, 45 sec, peak power 75, duty factor 10, cycles burst 200). The chromatin was sheared using Covaris E220 (900 sec, peak power 140, duty factor 5, cycle burst 200). The quality of the shearing was inspected on a Bioanalyzer 2100 (Agilent).  After sonication and 10 min high-speed centrifugation, fragmented chromatin was recovered in the supernatant. Chromatin was precleared by addition of 50 μl of Protein A-Agarose (PA) suspension (Roche 11134515001) followed by overnight incubation at 4°C. PA was removed by centrifugation, antibodies were added to the supernatant, and samples were incubated for 4 hr at 4°C in a rotating wheel. PA (50 μl) was added, and incubation was continued overnight at 4°C. Antibody-protein complexes were collected by centrifugation at 4,000 rpm for 1 min, and the supernatants were discarded. Samples were washed four times in Lysis buffer (with only 0.05% SDS) and twice in 1 mM EDTA, 10 mM Tris (pH 8) buffer (each wash, 5 min at 4°C). Chromatin was eluted from PA in 150 μl of 10 mM EDTA, 1% SDS, 50 mM Tris (pH 8) at 65°C for 15 min, followed by centrifugation and recovery of the supernatant. The eluate was incubated overnight at 65°C to reverse crosslinks and treated with Proteinase K for 3 hr at 50°C.  The chromatin was purified using ChIP DNA Clean & Concentrator columns (Zymo Research). In order to quantitatively assess the reduction of HP1 binding in the background of different mutants (HP1-KD and K9M) lambda DNA spike ins were added during the process. To perform a quantitative ChIP experiment all samples were processed in parallel starting from the same amount of embryos. After the preclearing a 20 µl aliquot of the sample was reverse crosslinked overnight at 65 °C and treated with RNAse and Proteinase K. The subsample was used to calculate the total amount of chromatin in the sample. All samples were then adjusted to contain the same amount of chromatin. Of the chromatin solution 10% were kept as an Input and lambda DNA was added at a final dilution of 1:1000 (usually around 60 pg of lambda DNA, when IPs were performed from 600 ng of total chromatin).  To account for differences in the amount of immunoprecipitated DNA 2 pg of lambda DNA per sample were added to the elution buffer. The lambda DNA was diluted in elution buffer and the same batch of buffer was used to elute all samples processed in parallel. libraries were prepared using NEB Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturers instructions