Total RNA was extracted and DNAse-I treated using a spin column-based RNA purification kit (Macherey–Nagel). Reverse transcription was performed with 500 ng of RNA using random primers and SuperScriptII (Invitrogen). Primers were designed using Primer 3 [84 (link)] and used for SYBER Green qPCR (Applied Biosystems). All primers sequences are listed in Additional file 9 : Table S1. For mRNA sequencing, 100-bp RNA-seq libraries were prepared using 200 ng of total RNA and the Illumina TruSeq Stranded mRNA reagents (Illumina). Three replicates from independent experiments for each sample for wild-type and DNMT TKO cells and two replicates for TET TKO cells were selected for high-throughput sequencing. Cluster generation was performed with the resulting libraries using the Illumina TruSeq SR Cluster Kit v4 reagents and sequenced on an Illumina HiSeq 2500 (Illumina).
Syber green qpcr
Manufactured by Thermo Fisher Scientific
SYBER Green qPCR is a real-time PCR reagent that utilizes SYBR Green I dye to detect and quantify DNA molecules. It provides a sensitive and reliable method for gene expression analysis, copy number determination, and other quantitative PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Truseq stranded mrna reagents, by Illumina (1 mentions)
Truseq sr cluster kit v4 reagents, by Illumina (1 mentions)
Spin column based rna purification kit, by Macherey-Nagel (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
2 protocols using syber green qpcr
1
Transcriptome Analysis of DNMT and TET Knockouts
2
ChIP-seq Protocol for Chromatin Immunoprecipitation
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Cells were harvested, washed with PBS, fixed in 2 ml fixation buffer (10 min in 1% formaldehyde, PBS), quenched with 10 ml 250 mM Tris–HCl pH 8, washed three times with PBS, pelleted, lysed by resuspension on ice in 1 ml sonication buffer (10 mM Tris pH 8, 1 mM EDTA, 0.2% SDS, protease inhibitors), transferred to TC 12 × 12 tubes (Covaris) and sonicated (Covaris settings: 20 min, 5% duty cycle, 140 W, 200 cycles). Sonication was assessed by reverse cross-linking (37°C RNAse A at 1 μg/μl 1 h, then 65°C, Proteinase K at 400 ng/μl, overnight), followed by DNA extraction. Fragment size (between 200 and 400 bp) was checked on a Bioanalyzer (Agilent 2100). Immunoprecipitations were performed in IP buffer (10 mM Tris at pH 8, 1 mM EDTA, 0.1% SDS, 150 mM NaCl, 10% Triton X-100 and protease inhibitors) overnight. Chromatin was reverse cross-linked (65°C, Proteinase K at 400 ng/μl, overnight) and DNA was further extracted for analysis. ChIP samples were used for SYBER Green qPCR (Applied Biosystems) or library preparation for sequencing as previously described (31 (link)).
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