Pbmal1 hr

Exon 8 of Bmal1 and exons 6 and 7 of Clock were deleted in β-TC6 cells by CRISPR–Cas9 and homology-directed repair (HDR). For Bmal1, intronic DNA flanking Bmal1 exon 8 was cloned into pTOPO2.1 (pBmal1-HR) (Invitrogen), and cells were cotransfected with guide RNA, Cas9 [pSpCas9(BB)-2A-Puro; Addgene], and pBmal1-HR plasmids. Stably integrated clones were selected for neomycin resistance (G418, Mediatech), and single colonies were hand picked and cultured individually. RNA and protein were extracted from these colonies and Bmal1 mRNA and protein were assessed by qPCR and Western blot. For Clock, cells were cotransfected with guide RNA, Cas9 (Clock CRISPR/Cas9 KO plasmids; Santa Cruz Biotechnology), and Clock HDR plasmids (Santa Cruz Biotechnology sc-419693-HDR). Stably integrated clones were selected for puromycin resistance (puromycin dihydrochloride, Sigma-Aldrich), and single colonies were hand picked and cultured individually. RNA and protein were extracted from these colonies and Clock mRNA and protein were assessed by qPCR and Western blot.