title: GSM8865846: Sample 9 Mouse Liver, Cold Exposure TFEB ChIP-Seq; Mus musculus; ChIP-Seq
library_name: GSM8865846
library_construction_protocol: 1. Weigh 100 mg of frozen tissue (25 mg/IP) on dry ice from each sample, place each sample into one well of a 6 well tissue culture dish on wet ice with 1 mL cold PBS + PIC (5 uL PIC (200x)/1mL PBS). Chop liver into small pieces. Transfer chopped liver (with 1mL pipette tip with tip cut off) and PBS to Eppendorf tubes.  2. Add 93.7 uL of 16% EM grade formaldehyde to make a final concentration of 1.5% and incubate at room temp for 10 minutes rotating.  3. Add 110 uL of 10 x glycine per 1mL of PBS + PIC and incubate at room temp for 5 minutes rotating.  4. Centrifuge at 500 g for 5 minutes at 4°C. Remove supernatant, resuspend in 1 mL cold PBS + PIC and spin down again at 500 g for 5 minutes at 4°C. Remove supernatant and resuspend in 1mL cold PBS + PIC  5. Transfer to the 2mL Dounce on ICE and Dounce with pestle A for 10 strokes, followed by pestle B for 20 strokes. Transfer samples back to Eppendorf tubes.  6. Spin samples at 2000g for 5 minutes at 4°C. SimpleChIP Plus Enzymatic Chromatin IP Kit was used according to manufacturer's instructions. Briefly, 100mg of tissue was used per mouse and two immunoprecipitations were performed with rabbit anti-TFEB antibody, and two immunoprecipitations were performed with normal rabbit IgG control. ChIP DNA was purified using Zymo ChIP DNA Clean and Concentrator Kit. Purified immunoprecipitated and input DNA was quantified and assessed for sheering using the Qubit dsDNA HS Assay Kit and Agilent DNA HS chip, respectively. Samples were prepared according the TruSeq ChIP Sample Preparation kit. Libraries were size selected for an average insert size of 350 bp using SPRI-based bead selection. Quality and quantity of the finished libraries were assessed using an Agilent Tapestation and Qubit dsDNA HS Assay Kit, respectively. Paired end 150bp sequencing was performed on the NovaSeq 6000 by the UW-Madison Biotechnology Center.
identifiers.namespace: GEO
identifiers.id: GSM8865846_r1