GSM8992153: ATAC_Homeostasis_rep2; Danio rerio; ATAC-seq
600 each embryos of the Tg (she:H2A-mCherry)psi56Tg lateral line marker line were separately dissociated in 4.5ml 0.25% Trypsin-EDTA (Gibco) for 3.5 minutes on ice by triturating with a 1ml pipette repeatedly. Supernatant containing cells was filtered through a 70μm Cup Filcon (BD Biosciences) and cells were pelleted by centrifugation at 720g for five minutes at 4C. Cells were washed in cold PBST by resuspending the pellet and pelleted again at 720g for five minutes at 4C. Cells were filtered through a 35μm strainer (Falcon-Corning), and stained with Draq5 (BioStatus) at a dilution of 1:500. Cells were then sorted on a BD Influx Cell Sorter (BD Biosciences). mCherry and Draq5 double positive cells were sorted into DPBS. Cells were pelleted by spinning in a microcentrifuge for 5 mins at 4°C and 1000g. Supernatant was removed carefully by pipette, and cells were resuspended in tagmentation reaction mix (22.5 μl H2O, 25μl TD Buffer (Illumina), and 2.5 μl TD Enzyme (Illumina)) and incubated in a ThermoMixer (Eppendorf) at 37°C for 30 minutes, 1000 RPM. 50 μl reverse crosslinking buffer (final concentration of 50mM TRIS pH 8, 1mM EDTA, 1% SDS, 200mM NaCl, 5ng/ml Proteinase K (Roche)) was added and mixture was incubated at 65°C overnight in a ThermoMixer. Tagmented DNA was purified with a DNA Clean & Concentrator 5 kit (Zymo Research) and eluted in 20 μl H2O. Library PCR amplification was performed with 20 μl eluted DNA, 25 μl NEBNext Ultra II Q5 Master Mix, 2.5ul forward primer Ad1 25M stock, 2.5ul reverse barcode primer115 Ad 2.x 25M stock. PCR conditions as follows: 72°C 5 mins, 98°C 30 seconds, 11 cycles of 98°C 10 seconds, 63°C 30 seconds, 72°C 1 minute, 4°C hold. Library was size selected with SPRIselect beads (Beckman Coulter) with the following conditions: 0.8x volume of beads first selection, the supernatant was retained and an additional 1.0x volume of beads was added (total volume 1.8x). The bead pellet was retained and rinsed with 85% EtOH, dried and DNA was eluted in 30ul H2O. Libraries were analyzed on a Bioanalyzer 2100 (Agilent) to evaluate tagmentation and quantified on a Qubit 3.0 (Thermo Fisher).