Takara ligation mix

Total RNA was extracted from cultured cells with an RNeasy Mini Kit (Qiagen). mRNA-Seq libraries were generated using an mRNA-Seq Sample Preparation Kit (Illumina) according to manufacturer's protocol with minor modifications. Poly(A)⁺ RNA was enriched from 1 μg total RNA by two successive rounds of oligo(dT) selection, fragmented and then used for first-strand cDNA synthesis by random hexamer priming. After second-strand cDNA synthesis, double-stranded DNA was repaired using T4 DNA polymerase, Klenow enzyme and T4 polynucleotide kinase (New England Biolabs), and then treated with Klenow exo- to add an adenine to the 3′ end. After ligation of the Index PE Adaptor oligo mix (Illumina) using Takara ligation mix (Takara), the adaptor-ligated DNA was amplified using primers, InPE2.0/1.0 and index (Illumina), by 18 cycles of PCR. The amplified libraries were isolated from an agarose gel. The DNA samples were purified using a QIAquick MinElute Kit (Qiagen) at each preparation step.

ChIP DNA and input DNA ends were repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs, Ipswich, MA, USA), followed by treatment with Klenow exo- to add an A base to the 3’-end. After ligation of the Genomic Adaptor Oligo Mix (Illumina, San Diego, CA, USA) using TaKaRa Ligation Mix (Takara Bio, Shiga, Japan), the adaptor-ligated DNA fragments were amplified with Paired-End Sample Prep Oligo primers (Illumina, San Diego, CA, USA) for 18 cycles. The amplified library was separated on a 2.0% agarose gel, and the samples were purified using the QIAquick MinElute kit (Qiagen, Hilden, Germany) after each preparation step. The purified library was used for cluster generation and sequencing analysis on a HiSeq 2000 (Illumina, San Diego, CA, USA). The raw Illumina sequencing data are available from the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) under accession number GSE63538.

Sequencing was performed using a Genome Analyzer IIx (Illumina). PolyA+ RNA was enriched from 100 ng of total RNA by two successive rounds of oligo(dT) selection. The polyA+ RNA was fragmented and first-strand cDNA was synthesized by random hexamer priming. Following second-strand cDNA synthesis, dsDNA was repaired using T4 DNA polymerase, Klenow enzyme, and T4 polynucleotide kinase (PNK) (New England Biolabs), followed by treatment with Klenow exo- to add an A base to the 3′ end. After ligation of the adaptor using TaKaRa Ligation Mix (TaKaRa), the adaptor-ligated DNA was amplified using PCR primers for 12 cycles, and the amplified library was isolated using the E-gel Electrophoresis System (Life Technologies). The samples were purified using a QIAquick MinElute Kit (Qiagen). Sequenced reads were mapped onto the mouse genome (mm9) using Tophat (version 2.0.10). Gene expression levels (FPKM; Fragments per kilobase of exon per million mapped sequence reads) were estimated by Cufflinks (version 2.1.1) using the parameters “-u -b (on cuffdiff)”. We classified genes into 11 groups based on the expression levels: Silent (FPKM = 0) and 10 groups with different expression levels separated at 10 percentile intervals (q0–10 %, q10–20 %, … and q90–100 % of genes from the lowest to the highest FPKM).
For a close look at ChIP-seq results, IGV_2.0.34 was used, as in Fig.4a, b.