GSM8946496: Mouse_Otx2_Atlas_Biological_Rep2_technical_Rep1; Mus musculus; OTHER
Nuclei were isolated according to the described protocol 75, using a lysis buffer modified to contain 0.01% Tween-20 and 0.01% Nonidet P40, as well as 1X protease inhibitor cocktail (Roche, 11873580001) in all solutions. Samples were lysed for 5 minutes in the lysis buffer with gentle trituration throughout, as described. CUT&RUN was performed using the CUTANA™ ChIC/CUT&RUN Kit (Epicypher, 14-1048). For each reaction, either 500,000 13LGS nuclei or 385,000 mouse nuclei were used. The CUTANA protocol v4.0 protocol was followed with minor modifications. In brief, isolated nuclei were resuspended in CUT&RUN wash buffer and added directly to Concanavalin A beads. Then 0.5 µg of the following antibodies were added to each respective reaction: IgG control (EpiCypher, 13-0042), H3K4me1 (EpiCypher, 13-0057), H3K4me3 (EpiCypher, 13-0041), H3K27ac (EpiCypher, 13-0059), H3K27me3 (EpiCypher, 13-0055), NeuroD1 (Cell Signaling, 62953), Otx2 (R&D Systems, BAF1979), or Otx2 (Atlas Antibodies, HPA000633).  H3K9me3 (Abcam, ab8898) was used at 0.41 µg of antibody per reaction. The total CUT&RUN DNA yield, up to 5 ng of total purified DNA, was used for library preparation, using reagents from CUTANA™ CUT&RUN Library Prep Kit (EpiCypher, 14-1001) or NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB, E7645S). The EpiCypher CUTANA Library Prep Kit User Manual v1.3 was followed and indexing performed using NEBNext® Multiplex Oligos for Illumina® (NEB, E7395S) or Dual Index Kit TT Set A (10X Genomics, PN-1000215). Final libraries were sequenced at the Novogene sequencing core (Sacramento, CA, USA) on the NovaSeq X Plus (Illumina) to generate paired-end 150 base pair reads.