GSM6122982: Retina 1 Cut&Run H3K9me3; Homo sapiens; OTHER
Peripheral retina samples were dissociated using papain as previously described (Fadl et al., 2020), cryopreserved in HBSS solution containing 10% DMSO and slowly frozen using a Mr. Frosty container (Invitrogen, CA, USA). CUT&RUN was performed as previously described (Skene & Henikoff, 2017) using 200-300,000 cells per experiment. Antibodies against H3K9me3 (Rabbit, cat.no. ab8898, Abcam, Cambridge, UK), H3K4me3 (Rabbit, cat.no. ab8580, Abcam, Cambridge, UK) and control IgG (Rabbit, cat.no. 011-000-002, Jackson ImmunoResearch Laboratories, PA, USA) were used at a concentration of 1:100 in 100ul and pA-MNase conjugated to protein A (generous gift of Dr. Steven Henikoff, Howard Hughes Medical Institute, WA, USA) was used at a concentration of 700 ng/ml. Released DNA fragments were purified using QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). Libraries were prepared with SMARTer® ThruPLEX® DNA-Seq Kit (Takara Bio USA, Inc, CA, USA) and amplified with 15 PCR cycles (60°C extension). Pair-end sequencing was performed with read length of 50 base pairs using the HiSeq 2500 platform (Illumina, CA, USA).