GSM6935744: control rep 2; Mus musculus; OTHER
For each biological sample, two freshly dissected retinas from one individual animal were pooled for and dissociated in PBS, 50 mM HEPES, 0.05 mg/ml DNase I and 0.025 mg/ml Liberase for 35 min with intermediate trituration. Cells were passed through a 40 μm nylon cell strainer, washed with washing buffer (1X PBS, 2% BSA, 0.1% sodium azide, 0.05% EDTA), and red blood cells were lysed in RBC lysis buffer. CUT&RUN was performed using a CUT&RUN Assay Kit (Cell Signaling 86652) according to manufacturer's protocol. 300K single cells were used for each reaction. 2 µl rabbit anti-H3K27me3 antibody (Millipore 07-449) was used for H3K27me3 immunoprecipitation. This antibody has been validated for specificity by ENCODE. 5 µl Rabbit I (DA1E) mAb IgG XP Isotype Control provided by the kit was used as a negative control. DNA fragments were purified on spin columns, eluted with 50 µl buffer (Cell Signaling 14209) and provided to University of Utah High Throughput Genomics Core for library generation. DNA quality was confirmed by PCR, libraries were created with the NEBNext UItra II DNA Library Prep Kit (New England Biolabs E7645S), and 150 bp paired-end sequencing was performed on a NovaSeq 6000 using the XP chemistry workflow (20021664) and S4 reagent Kit v1.5 (20028312).