ChIP-Seq for human brain: cerebellar cortex(CBC), H3K27me3: Sample HSB187_CBC_chromatin_H3K27me3
Tissues from the right and left hemispheres were pooled. Dissected tissue was pulverized in liquid nitrogen using a ceramic mortar and pestle and cross-linked with formaldehyde at a final concentration of 1% for 15 minutes at room temperature. Cross-linking was quenched by adding glycine to a final concentration of 125 mM and tissue was washed twice in ice-cold 1X Dulbecco’s PBS. Tissue was then dounced in ice-cold 1X-PBS and centrifuged to obtain a pellet of single cells. To isolate nuclei, the cell pellet was first incubated in Lysis buffer 1 on a rotator at 4 C, followed by incubation in Lysis buffer 2 at RT, with a centrifugation step after each incubation. Nuclei in second pellet were lysed by suspending in Lysis buffer 3. Chromatin was sonicated to sizes between 200 bp and 500 bp with Misonix 4000. For each ChIP reaction, 50ul of washed Dyna Protein G beads were incubated with 5 μl of the respective antibody for 12 h at 4 ˚C. Beads were washed thrice with ice-cold BSA solution, once with 1X TE, and then suspended in 50 μl of ice-cold BSA solution. 50 μg of the sheared chromatin was added to beads and the mixture was incubated for at least 16 h at 4 ˚C. Dynabeads were washed at room temperature eight times with RIPA buffer and once with 1X TE. Immunoprecipitated chromatin was eluted in 200 μl of Elution buffer at 65˚C for 15 minutes, with occasional vortexing. The eluted protein-chromatin complex was reverse cross-linked by overnight incubation at 65˚C. ChIPped and input DNA were treated with RNaseA, Proteinase K and purified with a Qiagen purification kit. For each sample, 5 ng of the purified immunoprecipitated DNA was used to prepare sequencing libraries. Sequencing libraries were generated using the ChIP-seq DNA sample prep kit (Illumina, IP-102-1001) according to the manufacturer’s protocol
alignment_software
bowtie 2.0