Agilent dna 1000 chip kit

Total RNA of cells was harvested with TRIzol (Takara, Terra Bella Ave, CA, USA) and quantified by a NanoDrop 2000. RNA integrity and gDNA contamination were tested by denaturing agarose gel electrophoresis (Thermo Fisher Scientific, Waltham, MA, USA), and the sequencing library was determined by an Agilent 2100 Bioanalyzer using an Agilent DNA 1000 chip kit (Agilent, Santa Clara, CA, USA). RNA-seq analyses were performed on HiSeq 4000 platforms (Illumina, San Diego, CA, USA). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis is a functional analysis mapping genes to KEGG pathways and was performed by DAVID^{29 (link)}. Heatmap was built using pheatmap in the R library.

To generate the library, the products of the (h)MeDIP, as well as the supernatant input DNA, were purified using Qiagen MinElute columns and eluted in 16 μl of elution buffer (Qiagen). Fourteen cycles of PCR were performed on 5 μl of the immunoprecipitated DNA using the single-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns; then, a final size selection (~300–600 bp) was performed using AMPure XP beads. Quality control of the libraries was performed by an Agilent 2100 Bioanalyzer using an Agilent DNA 1000 Chip Kit. An aliquot of each library was diluted in the elution buffer to 5 ng/μl, and 1 μl was used in real-time PCR reactions to confirm enrichment of the (hydroxy)methylated region.

The entire pituitary gland of each animal was homogenized in 1 mL of TRIzol reagent (Invitrogen, Karlsruhe, Germany). Total RNA was extracted in accordance with the manufacturing instructions. The extracted RNA was additionally treated with DNase I using the RNase-free DNase kit (Base-Zero DNase, Biozym, Hessisch Oldendorf, Germany) and with the Zymo^® RNA Clean&Concentrator kit (Zymo Research, Irvine, CA, USA). The RNA quality was assessed using an Agilent RNA 6000 Nano kit and 2100 Bioanalyzer (Agilent, Waldbronn, Germany). Samples with RNA integrity numbers (RIN) >8 [12 (link)] were used to generate a DNA library using a TruSeq Stranded mRNA Sample Preparation kit in accordance with the manufacture’s protocol (Illumina, Berlin, Germany). Essentially, polyadenylated mRNA molecules were enriched from 2 µg of total RNA using poly-T oligo-coated magnetic beads and chemically fragmented under elevated temperature conditions. The fragmented RNA was then reverse-transcribed into cDNA using random hexamers and Superscript II reverse transcriptase and ligated with TruSeq RNA adapters containing a unique DNA sequencing index to enable multiplexing. The DNA libraries were quality-control assessed for fragment-size distribution using an Agilent Technologies 2100 Bioanalyzer and Agilent DNA-1000 Chip kit.