GSM3003970: Liver_BMAL1_Input_replicate1; Mus musculus; ChIP-Seq
Mouse liver, kidneys, and heart were extracted were collected at ZT06 (middle of the light phase), rinsed in ice-cold 1X PBS, homogenized and crosslinked with 1% formaldehyde in 1X PBS at room temperature. Nuclei were then purified, sonicated and immunoprecipitated with BMAL1 antibody. BMAL1 and input ChIP-Seq libraries were generated from liver, kidney, and heart samples originating from the same animals (n = 3 mice per tissue) using NEBNext® ChIP-Seq Library Prep Master Mix Set (# E6240, NEB) as per the manufacturer's instructions. DNA from ChIP and input were quantified using a Quantus Fluorometer (# E6150, Promega), and 10 ng was used to generate the libraries. DNA end repair was performed with NEBNext End Repair Reaction Buffer and Enzyme Mix for 30 minutes at 20°C. dA-Tailing of end-repaired DNA was performed with NEBNext dA-Tailing Reaction Buffer and Klenow Fragment (3'-5' exo) for 30 minutes at 37°C. Adapter ligation of dA-tailed DNA was performed with Quick Ligation Reaction Buffer, NEBNext Adaptor (1.5 μM), and Quick T4 DNA Ligase. Libraries were generated by PCR amplification of adaptor ligated DNA using with NEBNext Multiplex oligonucleotides and Phusion Taq (M0530S). Libraries were amplified for 16 cycles. Libraries were quantified with qPCR with TRUseq library standards, and with a Quantus Fluorometer. DNA cleanup between each reaction was performed using Solid Phase Reversible Immobilization (SPRI) beads generated in the lab from Sera-mag SpeedBeads (# catalog number 09-981-123, Thermo-Fisher). BMAL1 ChIP-seq libraries were sequenced with an Illumina NextSeq with a sequence length of 75bp.
GEO Accession
GSM3003970