Double stranded cdna synthesis kit

In order to correlate HA/FLAG-H3.3 stability with the presence of histone modifications and/or gene expression levels, we constructed ChIP-Seq libraries for H2A.Z, H3K4me1, H3K4me3, H3K9ac, H3K27me3, and RNA-Seq libraries.
For RNA-Seq library construction, poly-adenylated RNA was isolated from 5 ug of total RNA isolated from undifferentiated ESCs cultured on gelatin-coated plates, using the Dynabeads mRNA Direct kit (Invitrogen). Double-stranded cDNA was generated with the Double-stranded cDNA synthesis kit (Invitrogen), sonicated, and subsequently processed exactly like ChIP DNA.
ChIP material was blunt-ended and phosphorylated with the End-it-Repair kit (EPICENTRE). Illumina genome sequencing adaptors were ligated with T4 DNA ligase (New England Biolabs) after addition of adenosine nucleotides, using exo-Klenow. Samples were PCR amplified with multiplexed Illumina genomic DNA sequencing primers. PCR products (250 to 450 bp in size) were gel purified and submitted for Illumina deep sequencing.

Transcriptome sequence were detected by Majorbio company. Briefly, the TRIzol reagent (Takara, Japan) was used to extract RNA from A549 tumor cells using the manufacturer’s instructions. Then Genomic DNA was removed using DNase I (Takara, Japan). The RNA was quantified using ND-2000 (NanoDrop Technologies, USA), and assayed for quality using a 2100 Bioanalyzer (Agilent, USA). The high-quality RNA samples (OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0>1 μg) were finally selected for the construction of sequencing libraries. After quality control, messenger RNA was first isolated and fragmented using oligo(dT) beads and fragmentation buffer, depending on the effective concentration and the amount of target ex vivo data required. Double-stranded cDNA was then synthesized, using a double-stranded cDNA synthesis kit (Invitrogen, USA) and Random Hexamer primers (Illumina, USA), followed by a library protocol including end repair, phosphorylation and "A" base addition. cDNA target fragments were then PCR amplified. A read length of 200–300 bp was selected. The unprocessed tail reads were then trimmed and quality controlled using SeqPrep and sickle. The clean reads were separately mapped to the reference genome using HISAT2 software. The mapped reads of each sample were assembled by StringTie.