title: GSM8839525: 25mM glucose, H3K18Ac (CUT&Tag), replicate 2; Mus musculus; OTHER
library_construction_protocol: CUT&Tag protocol was performed using CUT&Tag-IT® Assay Kit – Tissue (Active Motif) with slight modifications. In brief, explanted retinas were dissected and cells were dissociated in a 5 ml polypropylene round-bottom tube with a papain dissociation protocol adapted from previous study {Fadl, 2020 #16} . After dissociation the cells were washed using 1X wash buffer provided in the kit at 200g for 5 minutes. CUT&Tag was performed using 500,000 cells per experiment following kit protocol. Antibodies against H3K18La (Rabbit, cat.no. PTM1406), and H3K9me3 (Rabbit, cat.no. ab6002, Abcam, Cambridge, UK) were used at a concentration of 1:100 in 100 μl and pA-MNase conjugated to protein A (generous gift of Dr. Steven Henikoff, Howard Hughes Medical Institute, Washington, USA) used at a concentration of 700 ng/ml. Total RNA from explant retinae was extracted using TRIzol® (Invitrogen, Carlsbad, CA), treated with DNase and cleaned up using the MagMAX mirVana Total RNA Isolation Kit (Applied Biosystems, Foster City, CA) following the manufacturer's instructions. Libraries were constructed with SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian (Takara Bio USA, Mountain View, CA) with 4 ng of RNA and 13 PCR cycles library amplification. Paired-end sequencing of 101 bases were obtained using the NextSeq 2000 (Illumina, San Diego, CA). CUT&Tag-IT® Assay Kit – Tissue (Active Motif)
identifiers.namespace: GEO
identifiers.id: GSM8839525_r1