7900ht real time pcr machine

Total RNA was isolated using the ZR-96 Quick-RNA kit (Zymo Research, R1052), which includes an on-column DNase I treatment step, and eluted in 30 μL RNase-free dH₂O. cDNA was prepared using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher #4368814). For detection of transcripts from liver genes in fibroblast heterochromatin, TaqMan-based quantitative PCR was performed using the TaqMan Gene Expression Master Mix (Thermo Fisher #4369016), and data was normalized to GAPDH (for siRNA screen in Figure 6, S5C), or an average of GAPDH and 18SRNA endogenous controls (all other experiments). See Key Resources Table for TaqMan primers/probes used. For verification of siRNA knockdown efficiency, qPCR was performed using Power SYBR Green PCR Master Mix (Thermo Fisher #4367659), and data was normalized using the GAPDH primer as an endogenous control (see primers listed below). Both SYBR and TaqMan qPCR reactions were run in 384-well format on an 7900HT Real-Time PCR machine (Thermo Fisher #4329001), using the following thermal cycler protocol: 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15 s then 60°C for 1 min. For SYBR-based qPCR reactions, a dissociation curve was generated to verify that a single PCR product was generated.
Primers for SYBR-based RT-PCR experiments to verify knockdown efficiency:

Purified DNA samples from gradient fractions were quantified with Quant-iT PicoGreen dsDNA Assay (Thermo Fisher P7589) and diluted with TE buffer to 0.1 ng/μl, to allow loading of equal DNA mass per qPCR reaction. DNA concentrations were verified after dilution by repeat PicoGreen assay and were adjusted as needed. 10 μl qPCR reactions were prepared in 384-well optical plates with 2 μl DNA sample, 0.1 μl primer mix (10μM each primer), and 5 μL Power SYBR Green PCR Master Mix (Thermo Fisher #4367659). Plates were in a 7900HT Real-Time PCR machine (Thermo Fisher #4329001), using the following thermal cycler protocol: 50°C for 4 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s then 54°C for 15 s then 72°C for 45 s, with a dissociation curve generated to verify that a single PCR product was amplified. qPCR results for each fraction were normalized to the input sample.
Primer sequences for detecting sites inside and outside DBRs were from (Soufi et al., 2012 (link)) and are listed below. All PCR amplicons correspond to Oct4/Sox2 binding sites: bound in fibroblasts and ES cells for non-DBR sites, and bound only in ES cells for DBR sites (Soufi et al., 2012 (link)). There is also enrichment for H3K9me3 at the DBR sites but not the non-DBR sites by ChIP in fibroblasts (Soufi et al., 2012 (link)).

Total RNA was isolated using the ZR-96 Quick-RNA kit (Zymo Research R1052), this includes an on-column DNAse I digestion. The samples were then eluted in 30 μL RNAseq Free ddH2O. cDNA was prepared using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher 4368814). Primers were designed for each transcript targeted by an siRNA (Supplementary Table 2). For assessing the expression of pre-RNAs primers were designed with one primer targeting an intron. qPCR was performed using Power SYBR Green PCR Master Mix (Thermo Fisher 4367659), and data were normalized using the GAPDH primer as an endogenous control (Supplementary Table 2). qPCR reactions were run in 384-well format on an 7900HT Real-Time PCR machine (Thermo Fisher 4329001), using the following thermal cycler protocol: 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15s then 60°C for 1 min. For SYBR-based qPCR reactions, a dissociation curve was generated to verify that a single PCR product was generated.