title: GSM9207702: KGMKWT2MG; Mus musculus; RNA-Seq
library_construction_protocol: neural retinas (n = 4 per pooled sample) were dissected free of retinal pigment epithelium. Retinal tissue was dissociated into single cells via a papain dissociation system (Worthington, Columbus, OH), passed through a 40 µm cell strainer, and washed with PBS containing 0.5% bovine serum albumin (BSA). Dead cells and debris were depleted with the Dead Cell Removal Kit (Miltenyi Biotech, Bergisch Gladbach, Germany). The remaining cell suspension was incubated on ice for 10 min  with 15 µL of anti-GLAST (ACSA-1)-APC antibody at a 1:50 dilution (Miltenyi Biotech) protected from light. After washing, the cells were centrifuged (300g , 5 min), resuspended in 90 µL of MACS buffer and 10 µL of anti-APC microbeads (Miltenyi Biotech), and then incubated at 4°C for 15 min. Following a wash, the cells were passed through an LS column (Miltenyi Biotech) in a QuadroMACS separator. Unlabeled (GLAST⁻) cells were collected as flow-through; the column was washed 3x with 3 mL of MACS buffer. GLAST⁺ Müller glia were eluted with 5 mL of MACS buffer via a plunger. Total RNA from enriched Müller glia was extracted via the Arcturus™ PicoPure RNA Isolation Kit (Thermo Fisher, Waltham, MA). RNA quality was assessed via an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), and samples with an RNA integrity number (RIN) of 7.0 were used for library construction. cDNA was synthesized and amplified via the SMARTer v4 Ultra-Low Input RNA Kit (Clontech, Mountain View, CA). cDNA was fragmented on a Diagenode Bioruptor Pico (Diagenode, Denville, NJ), and sequencing libraries were prepared with the KAPA HyperPrep DNA Kit (Roche, Basel, Switzerland). Libraries were pooled and sequenced on an Illumina NovaSeq 6000 S4 flow cell (QB3 Genomics Core, UC Berkeley; RRID: SCR_022170) to generate 150 bp paired-end reads.