title: GSM3682453: PFC_mix_H3K79me2_1k_R1; Mus musculus; ChIP-Seq
library_construction_protocol: 50 mg of the frozen mouse prefrontal cortex tissue was placed in a glass dounce tissue grinder, add 5 ml of nuclei extraction buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 0.1 mM EDTA, 10 mM Tris-HCl and 0.1% Triton X-100), 50 µl of PIC, 5 µl of 100mM PMSF and 5 µl of 1M DTT, and place the grinder on ice. Once the tissue thaws, dounce the tissue with pestle A (D9063, Sigma-Aldrich) for 15 times, followed by douncing with pestle B (D9063, Sigma-Aldrich) for 25 times. Filter the homogenate through a 40-µm cell strainer into a new 15 ml centrifuge tube. Centrifuge the tube at 1000 g for 10 min at 4 °C. Discard the supernatant and gently resuspend the crude nuclei pellet in 500 µl nuclei extraction buffer. Add 5 µl of PIC, 0.5 µl of 100 mM PMSF, and 0.5 µl of 1 M DTT to the nuclei suspension. Transfer the nuclei suspension into a 1.5 mL Eppendorf microcentrifuge tube. Place the tube on ice. Add 750 µl of 50% iodixanol solution, 7.5 µl of PIC, 0.75 µl of 100 mM PMSF and 0.75 µl of 1 M DTT to the tube. Mix the solution well. Centrifuge at 10,000 g for 20 min at 4 °C. Carefully remove all the supernatant. Add 50 µl of PBS, 0.5 µl of PIC, 0.5 µl of 100 mM PMSF to the tube. Incubate on ice for 10 min. Resuspend the nuclei pellet by pipetting up and down gently. Check nuclei concentration with a hemocytometer to get 1000 nuclei per assay. Usually we can get ~150K nuclei after this   protocol. Library preparation was conduct using the Swift Bio S2 library preparation kit (Swift Biosciences) using ChIP DNA from 1000 nuclei (we can get ~150 pg DNA per assay). Use 2.5 μl of 20x EvaGreen  and 7.5 μl of the low EDTA TE buffer in the 50 μl amplification cycle reaction mix, instead of 10 μl of low EDTA TE buffer and amplification was stopped after samples saw an >3000 RFU increase (BioRad CFX Connect). Amplified DNA was then eluted into 10 μl low EDTA TE buffer where 1 μl could be used for qPCR analysis for preliminary quality control analysis, Kappa DNA quantification, and Tapestation fragment size analysis and the other 5 μl for library pooling. Libraries were pooled at 10 nM for sequencing by Illumina HiSeq 4000 with single-end 50 nt read.
attributes.tag: GEO Accession
attributes.value: GSM3682453