GSM5061623: control_rna_r2; Mus musculus; RNA-Seq
ChIP-Seq: Liver tissue (∼300 mg) was freshly excised from adult male KLF153xFLAG/WT mice. The ChIP-seq replicates reported in our study are derived from two separate pieces of liver tissue which came from the same mouse.  Liver tissue was minced, crushed, and fixed with 1.1% (wt/vol) formaldehyde for 10 min.  For chromatin preparation, fixed tissue was homogenized in lysis buffer 1 (50mM Hepes pH 7.5, 140mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitor cocktail) using a Dounce homogenizer, then rotated at 4°C for 10 min. Pellet was spun down and resuspended in lysis buffer 2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1 mM EDTA, 0.5mM EGTA, protease inhibitor cocktail) for 10 min at 4°C. The pellet was spun down and resuspend pellet in 1 ml sonication buffer (50mM Hepes pH 7.5, 140mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, protease inhibitor cocktail). The sonication buffer with chromatin was then sheared on a BioRuptor (Diagenode, Bioruptor® Plus, UCD-300) with samples continuously cooled at 4°C. The BioRuptor settings used were 30 sec on, 30 sec off with output setting on “high” for a total of 12 cycles. The sonicated chromatin was immunoprecipitated overnight at 4°C using 5g anti-FLAG (Sigma F1804) monoclonal antibody bound Dynabeads (Invitrogen 10004D) followed by extensive washing and elution. Input and immunoprecipitated chromatin were then reverse-cross-linked followed by purification of genomic DNA, which was then subject to library preparation and next generation sequencing. RNA-Seq: For RNA isolation from mouse tissues, tissue samples were disrupted/homogenized in TRIzol (Thermo Fisher) in a TissueLyser (Qiagen) using stainless steel beads (QIAGEN) at 30 Hz for a total of 4 min. Total RNA was isolated using Aurum Total RNA Fatty and Fibrous Tissue kit (Bio-Rad, 7326870). ATAC-seq: Primary hepatocytes were isolated from liver tissues of KLF15-LKO mice and Klf15flox/flox control mice. For each sample, 50,000 cells were pelleted after washing once with 1ml PBS. Cell pellets were then resuspended in 50mL cold lysis buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween-20, and 0.01% Digitonin) and incubated on ice for 3 min. 1ml cold wash buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20) was then added to the lysis. The sample was centrifuged at 500g for 10 min at 4°C. The pelleted nuclei were resuspended in 50mL transposition mixture [25mL 2X Tagment DNA buffer, 5mL of Tagment DNA Enzyme (Nextera Sample Prep Kit from Illumina, FC-121-1030, 16.5ml PBS, 0.5ml 0.1% digitonin, 0.5ml 10% Tween-20, 2.5ml H2O], and incubated at 37°C for 2hr in a thermomixer with 1000 rpm mixing. Transposed DNA was purified with MinElute Reaction Cleanup Kit (Qiagen, 28204) and eluted in 21mL water ChIP-Seq: Purified DNA were quantified using Qubit fluorometric analysis and assessed for quality using Agilent Bioanalyzer Nano DNA Chip. Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit from Illumina (NEB, E7645). Libraries were sequenced on a NextSeq 500/550 at the Genomics Core of The Gladstone Institutes (paired-end, 75 base pairs, >40M reads per sample). RNA-Seq: RNA-seq libraries were prepared with the Ovation Solo RNA-Seq kit (NuGEN). The final libraries were analyzed by Agilent Bioanalyzer Nano RNA Chip and quantified by KAPA qPCR. The library was then sequenced on an Illumina HiSeq 2500 at the Genomics Core of Gladstone Institutes. ATAC-Seq: Purified DNA was amplified using 25mL of NEBNext High Fidelity 2x PCR Master Mix, 1.25 mM Nextera custom primers with unique barcodes, and nuclease-free water. Samples were amplified using the following PCR conditions: 72°C for 5 min; 98°C for 30 s; and cycled at 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. Half of each sample was amplified for 12 cycles, purified, and assessed by Bioanalyzer (Agilent) for library quality. Sample concentration was quantified by Qubit (Invitrogen) prior to pooling. Pooled samples were sequenced on a NextSeq 500/550 at the Genomics Core of Gladstone Institutes (paired-end, 75 base pairs, >80 M reads per sample)