GSM5706762: Mphosph8Dn H3K9me3 replicate 1 [66592_M8KO_H3K9me3]; Mus musculus; ChIP-Seq
Subsequently, nuclei were isolated in nuclear isolation buffer (10 mM Tris-HCl, 50 mM sodium-disulfite, 1% Triton X-100, 10 mM MgCl2, 8.6% sucrose, pH6.5) and centrifuged 5 minutes at 455 g at 4°C. The resulting pellet was washed four times with 900 µl nuclear isolation buffer with centrifugation at 455 g at 4°C. The pelleted nuclei were then resuspended in 1.4 ml of PBS, filtered through a cell strainer and stained with a 1:1000 dilution of the anti-NeuN antibody (clone A60, Alexa Fluor 488 conjugated, Millipore MAB377X) for 45 minutes by rotation in the dark at 4°C after adding BSA to a final concentration of 0.1%. After washing in PBS, the stained nuclei were filtered through FACS tubes and sorted on a FACS Aria III (BD) sorter in PBS. After the FACS-sort, the pelleted nuclei were immediately resuspended in ChIP lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 1% SDS, pH 8.1). Equal amounts of Bioruptor-sonicated chromatin were diluted 10-fold and precipitated overnight with the H3K9me3 (Abcam ab8898) antibody. Input DNA was quantified using Quant-iT PicoGreen dsDNA Assay (Thermo Fisher). 1 ng of input DNA was used for library preparation with the Ultra Low Input Library Kit (Qiagen, 180497). The procedure of library preparation was done according to the manual provided by Qiagen with adapter dilution of 1:100. Quality control of the libraries included a fragment analyzer run and qPCR to determine average size and precise concentration. Based on the qPCR values, samples were pooled equimolarly to a concentration of 2.5 nM. The pool was loaded on an Illumina HiSeq2000 flowcell with a concentration of 18 pM and sequenced 50 bp single end.