GSM8266074: SAC, P16ZE12, n/a, rep315; Mus musculus; RNA-Seq
Retinas from ChatCre/+; ROSA-LSL-TD-Tomato mice were isolated at P1, P4, P8, P11 and P16 in ice cold HBSS, and the retinal pigmented epithelium and inner limiting membranes were removed. Retinas were digested with papain, triturated slowly 15-20 time with a P1000 pipet tip, strained into a 50 mL conical tube, and pelleted for 3 minutes at 1.3k rpm. The pellet was gently resuspended and cells were washed three times in ice cold Hibernate A media lacking Ca2+, Mg2+, and phenol red, with pelleting between the washes. Care was taken to tap the tube gently to dislodge the pellet before adding media and resuspending the pellet, to prevent cell lysis and subsequent accumulation of DNA and cellular debris, which forms as a sticky gel-like substance in the supernatant. Washed and resuspended cells in ice cold Hibernate A were subjected to FACS to isolate single cells in cell lysis buffer before proceeding with Smart-seq2 mRNA sequencing as in Chevee et al., 2018. A modified Smart-seq2 mRNA sequencing protocol was used to construct libraries, described in Chevee et al., 2018.