Sybr green supermix low rox

Total RNA was extracted using an RNeasy Mini Kit (Qiagen #74104). In brief, cells were lysed in TRIzol reagent followed by chloroform extraction and ethanol precipitation. Reverse transcription and qPCR were performed using a High Capacity cDNA reverse transcription kit (Applied Biosystems #4368814) with 2 µg per 40 μL reaction. All primer sequences are listed in Supplementary Table 3. Data was collected and analyzed using SYBR Green SuperMix Low ROX (Quanta bio # 95056-02k) with a ViiA 7 Real-Time PCR instrument (Applied Biosystems).

The human and mouse CBS gene was downloaded with the Ensembl genome browser with 10,000 base pairs before the transcription start site^{35 (link)}. The gene was analyzed for HSF1 consensus binding sites^{36 (link)}. Perfect HSF1 consensus binding sites or slight mismatches were found in the first exon and 3’UTR of both human and mouse CBS genes. Chromatin immunoprecipitation (ChIP) assays were performed as described previously^{72 (link)}. C4-2 cell chromatin was crosslinked for 10 min at room temperature with 1% formaldehyde added to cell culture medium. Cell lysates were sonicated (Branson 250 #102 C) for 3 cycles of 10 seconds. Protein G-conjugated agarose beads (Santa Cruz sc-2003) were used to immunoprecipitate chromatin complexes. Four μg of ChIP grade antibodies raised against HSF1 (Abcam EP1710Y #ab52757) or the HA tag (Abcam #ab9110) on the deactivated Cas9 were used to precipitate the chromatin. After de-crosslinking and isolation of DNA with a QIAquick PCR Purification Kit, samples were analyzed with quantitative PCR was employed on genomic DNA targets relative to input with SYBR Green SuperMix Low ROX (Quanta bio # 95056-02k) and a ViiA 7 Real-Time PCR instrument (Applied Biosystems). Primers used for ChIP-qPCR are listed in Supplementary Table 3. Control IgG ChIP-qPCR assays showed low background binding to binding sites.