Transcriptome analysis of Casz1 conditional mutant progenitors at postnatal day 2

Transcriptome analysis of Casz1 was performed using litters of postnatal day 2 retinas from crosses between Casz1Flox/Flox; R26-Stop-EYFP and Casz1Flox/Flox; IKCre-A; R26-Stop-EYFP. The IK-A Cre driver is described in Tarchini et al., Dev Dyn 241(12):1973-85. EYFP marks Cre expressing (and therefore cKO) cells. Cells were incubated for 30 min in RGM serum-free medium described in Cayouette et al., Neuron 40(5):897, supplemented with Hoechst 33342 (Invitrogen), and 4N cells were sorted using a MOFLO cytometer (Beckman) based on Hoechst, GFP expression, and propidium iodide exclusion. Cells were sorted directly into lysis buffer on ice and RNA was immediately extracted using RNeasy mini columns (Qiagen). Two independent experiments were performed, and the RNA samples were processed in parallel. RNA amplification was performed using Truseq PE Cluster kit v3 PE50 (Illumina). Deep sequencing was performed using Truseq stranded mRNA (Illumina) with mRNA enrichment and strand-specific parameters, using a Hiseq 2000 instrument (Illumina).