title: H3K27me2-profiling in autotrophic Chlorella sorokiniana by ChIP-Seq, 2nd rep
library_construction_protocol: Chlorella sorokiniana UTEX1230 cells (9 x 10^8 cells) were crosslinked at room temperature using 0.37% formaldehyde for 10 minutes, after which crosslinking was quenched by 0.125 M glycine for 10 minutes at room temperature. Cells were washed twice with water and centrifuged at 3000 g for 5 min at 4°C, subsequently resuspended in 200µl of Lysis Buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8, 1x cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche #04693132001)) and frozen with liquid nitrogen. Next, cells were sonicated using Bioruptor Plus with 20 cycles of 30 sec ON/OFF at 4°C to achieve DNA fragments size of approximately 200 bp and centrifuged for 5 min at 4500 g at 4°C to remove cell debris. The supernatant was diluted ten times with ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8, 167 mM NaCl, cOmplete™, EDTA-free Protease Inhibitor Cocktail (Roche #04693132001)), 200µl aliquot was used for each IP incubated overnight at 4°C with 1.5µg of antibody. Antibody specific for the following epitope was used: H3K27me2 (Abcam, ab24684, lot GR3390336-1). The next day, the sample was mixed with 20 µl Dynabeads Protein A (Thermo) and incubated for another 2 hours at 4°C. Next, beads were successively washed three times (5 min/wash) with Low Salt Wash Buffer (150 mM NaCl, 0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris-HCl pH 8), High Salt Buffer (500 mM NaCl, 0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris-HCl pH 8), LiCl Wash Buffer (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8) and finally with TE Buffer (10 mM Tris-HCl pH 8, 1 mM EDTA).  DNA was decrosslinked, purified, and eluted using IPureKit (Diagenode). Sequencing libraries were made with 5ng DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB #E7103S/L) following the manufacturer's instructions.