title: GSM6424704: Multiome_mutant_nr5a2_GFP_2.5dpf_head (snATAC-seq); Danio rerio; ATAC-seq
library_construction_protocol: Zebrafish embryos are screened for nr5a2:mGFP-DBD-del+ / scxa:mCherry+ and fin tips are collected for PCR-genotyping of oz3 allele (mutant) and wild-type allele (control) before 2 dpf. Control and mutant heads were decapitated between the eye and ear after anesthesia at 2.5 dpf. Dissected heads (twenty heads pre tube) were washed twice with fresh and iced Ringer's solution (116mM NaCl, 2.6mM KCl, 5mM HEPES, pH 7.0), followed by dissociation at 28.5 °C for 40 min by mechanical - nutating and pipetting every 5 min and enzymatic - in pre-warmed protease solution (0.25% trypsin, 1 mM EDTA, pH 8.0, and 20 mg/mL Collagenase D (from stock of 400 mg/mL Collagenase D in HBSS) in PBS) until fully dissociated. Dissociation reaction is stopped by 6X stop solution (6 mM CaCl2 and 30% fetal bovine serum (FBS) in PBS). Dissociated cells were collected by centrifugation for 5 min, 2000 rpm at 4°C and washed by suspension solution (1% FBS, 0.8mM CaCl2, 50U/ml Penicillin, 0.05mg/ml Streptomycin in Leibovitz Medium) twice and filtered by cell strainer before sorting. Live cells were fluorescence-activated cell sorted (FACS) to isolate GFP+ and exclude the cytoplasmic stain Zombie Violet (control) or nuclear stain DAPI (mutant) into 0.04% BSA/ PBS at 4°C. Nuclei isolation was performed per manufacturer's instructions (10X Genomics, protocol CG000169, low cell input protocol) with optimalization for zebrafish mesenchyme. FACS cells were pelleted for 15 min, 300 rcf at 4oC, and incubated with lysis buffer for 100 s on ice. Isolated nuclei were washed by Wash buffer and Nuclei buffer and checked for nucleus integrity under a fluorescence confocal microscope with DAPI staining before subjected to library construction. To capture accessible chromatin and transcripts from the same cells per manufacturer's instructions, accessible chromatin regions from isolated nuclei were first targeted and tagged by transposase. Tagged-chromatin and ployA mRNA from the same nuclei were pulled down and barcoded with the same sequences within isolated GEMs to achieve single-nuclei separation. Chromatin fragments were further indexed with sample-specific i7 indexes and linked with Illumina P5 and P7 sequences. Reverse transcription was performed in GEMs to synthesize cDNA from polyA mRNA. cDNAs were further fragmented and indexed with sample-specific i7 and i5 indexes and linked with Illumina P5 and P7 sequences. QC of libraries were checked with 4200 TapeStation system and Qubit dsDNA HS assay kit.