title: GSM7026011: P21_SC_WGBS_rep2; Mus musculus; Bisulfite-Seq
library_construction_protocol: Isolate cochlear sensory epithelium: For E13.5, the inner ear was dissected from the mouse head, followed by incubation in PBS containing 0.5 U/ml of Dispase II (Sigma D4693) and 275 U/ml of Collagenase Type 1 (Worthington LS004196) at room temperature for 10 minutes. The Dispase II and Collagenase were removed and quenched with PBS + 5% Fetal Bovine Serum (FBS) (Genclone 25-514).  The cochlear epithelium was separated from the mesenchyme and dissociated into single cells. For P1, P6, P8, and P21 cochleas, the inner ear was extracted and placed in a dish containing ice-cold modified artificial cerebrospinal fluid solution (ACSF) [119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 1 mM NaH2PO4, 26.2 mM NaHCO3, 10mM HEPES, 11mM D-Glucose] on top of an ice block. Cartilage and bone were removed to expose the cochlear epithelium. Finally, the cochlear epithelium was separated from the spiral ganglion and modiolus, and then placed into a separate dish containing ACSF on ice until all ears were processed.    Obtain single cell/nuclei: For E13.5 and P1, cochleas were enzymatically dissociated with a cocktail of 400µl of 0.25% Trypsin, 50µl of 1 mg/ml Dispase, and 50µl of 1 mg/ml collagenase for 20 minutes at 37°C. After incubation, the digested tissue was titurated 100 times using a small-bore 200µl pipette and centrifuged at 500x g for 5 minutes at 4°C. For P6, P8, and P21, the sensory epithelium and stria were placed into a 1ml Dounce tissue grinder (Wheaton 357538) on ice with 1 ml of chilled homogenization buffer (250mM sucrose, 25mM KCl, 5mM MgCl2, 20 mM HEPES-KOH pH 7.9, 20% glycerol). Before use, the following components were added at their respective concentrations: 1mM DTT (Thermo Scientific R0861), 0.5mM Spermidine (Sigma S0266), 1% BSA (Sigma A4503), and 1x cOmplete EDTA-free protease inhibitor cocktail (Roche 11836170001). NP-40 (Calbiochem 492015) was spiked into the Dounce tissue grinder at a final concentration of 0.3%. The tissue was homogenized with a chilled loose pestle by first pressing the pestle down and turning to grind the tissue, followed by quick upstrokes 10 to 15 times to create cavitation. It was critical to not lift the Dounce pestle above the liquid layer to reduce bubbles. This process was repeated with a chilled tight pestle. Although some epithelial tissue was sometimes still visible, no further homogenization was performed as this may jeopardize the integrity of the already-released nuclei. The homogenate was filtered using a Falcon 5ml round bottom polystyrene test tube with a 40 µm cell strainer snap cap and spun down at 1000 x g and 4°C for 10 minutes. The supernatant was decanted, and the pellet was resuspended in 1ml of nuclei wash buffer (NWB) (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.1% NP-40). Before use, the following components were added at the respective concentrations: 1mM DTT, 0.5mM Spermidine, 2% BSA, and 1x cOmplete EDTA-free protease inhibitor cocktail.     Purifying celltypes: Fluorescence-activated cell sorting (FACS) of cells or nuclei was performed on either an Aria I, Aria II, or FACSymphony (BD Biosciences). For CUT&RUN and CUT&Tag, cells or nuclei were sorted into NWB. For WGBS, cells or nuclei were sorted into lysis buffer (10 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, and 0.01% Triton X-100). For Single Cell Multiome ATAC + Gene expression (10x Genomics), cells or nuclei were sorted into PBS and 0.1% BSA. Cells/nuclei were used for downstream NGS experiments (WGBS, CUT&Tag, 10x Genomics scMultiome). WGBS: Cells/nuclei were lysed in lysis buffer (10mM Tris pH 7.5, 100mM NaCl, 1mM EDTA, and 0.01% Triton X-100) and digested with Proteinase K at 3mAU/ml at 65C for at least 1 hour. gDNA was purified using Genomic DNA Clean & Concentrator-10 (Zymo Research). Unmethylated Lambda DNA was spiked in at 0.5% of input gDNA. Libraries were prepared using the Pico Methyl-Seq Library Prep Kit (Zymo Research)  CUT&Tag: The following antibodies were used: Rabbit anti-H3K4me1 antibody (Active Motif 61633), rabbit anti-H3K4me2 antibody (Epicypher 13-0027), rabbit anti-H3K4me3 antibody (Active Motif 39159), rabbit anti-H3K27me3 antibody (Active Motif 39155). The guinea pig anti-Rabbit IgG antibody (Antibodies-online.com ABIN101961) was used as the secondary antibody. Tn5-tagmented DNA fragments were amplified using NEBNext HiFi 2x PCR Master Mix, a universal i5 primer and a uniquely barcoded i7 primer for 14 cycles. Library concentration and fragment distribution were measured on the Agilent Tapestation using a High Sensitivity D5000 ScreenTape (Agilent 5067-5592). Libraries were size-selected for DNA fragments greater than 150 bp using SPRISelect beads (Beckman Coulter B23318).    scMultiome  ATAC + Gene Expression protocol (10x Genomics)