title: GSM6131901: P0_IgG_3; Mus musculus; OTHER
library_construction_protocol: Single cells were obtained from female littermates. Freshly dissected retinas were pooled and dissociated in PBS, 50 mM HEPES, 0.05 mg/ml DNase I (Sigma D4513) and 0.025 mg/ml Liberase (Sigma 5401119001) for 35 min with intermediate trituration. Cells were passed through a 70 mm 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 (eBioscience 00-4333-57). Cell counts were determined using a Countess (Invitrogen C10228), and cells were split into technical replicates before performing CUT&RUN. P0 H3K27me3 and P0 IgG samples were run in technical triplicate, E14.5 H3K27me3 was run in duplicate, and E14.5 IgG was a single sample. CUT&RUN was performed using a CUT&RUN Assay Kit (Cell Signaling 86652) according to manufacturer's protocol. 2 µl rabbit anti-H3K27me3 antibody was used for H3K27me3 Immunoprecipitation and 5 µl Rabbit I (DA1E) mAb IgG XP Isotype Control provided by the kit was used as a negative control. Samples were purified on DNA spin column (cell signaling #14209) eluted with 35ul elution buffer. 5ul was used for PCR to validate sample quality and the rest used for library preparation. PCR showed specific bands with positive control primers for H3K27me3. DNA was 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 with the NovaSeq S4 Reagent Kit v1.5.