Bioanalyzer with high sensitivity dna chips

ChIP-seq libraries were constructed using a TruSeq ChIP Sample prep kit (Illumina) following the manufacturer’s instruction. In brief, 50 μl of ChIP DNA was end-repaired, 3′-adenyl tailed, and then ligated with different index adaptors. The adaptor-ligated DNA was size selected (250–300 bp) using agarose gel electrophoresis, followed by PCR amplification. The amplified DNA was purified with AMPure XP beads (Beckman Coulter). The quality of the ChIP-seq libraries was checked using a Bioanalyzer with high-sensitivity DNA chips (Agilent). ChIP-seq libraries were pooled and sequenced using an Illumina NextSeq platform with single-end reads of 76 bases.

cDNA was synthesized and amplified from the poly-A containing RNA transcripts in the single-cell lysate using the Smart-Seq2 method [35 (link)]. cDNA from cells at 4-cell stage was amplified by 15 cycles of PCR, while 16 cycles of PCR was applied on the cDNA from cells of 8-cell and 16-cell stage embryos. The amplified cDNA samples were cleaned up with Ampure XP beads (Beckman Coulter, CA, USA), quantified by Qubit fluorometer (Invitrogen, CA, USA), and examined on Bioanalyzer with High Sensitivity DNA chips (Agilent, CA, USA) for size distribution. Nextera DNA library prep kit (Illumina, CA, USA) was used to convert the cDNA samples to Illumina sequencing libraries. The libraries were pooled at equal molar amount and sequenced on Illumina HiSeq 2500 Rapid flowcells as single-end 75 nt reads with dual index reads following the standard Illumina protocol. Raw sequencing reads were filtered by Illumina HiSeq Control Software and only Pass-Filter (PF) reads were used for further analysis.