title: GSM2333632: Cux2_L2/3_500cell_rep1; Mus musculus; OTHER
library_construction_protocol: We used an isoflurane chamber to anesthetize adult male mice (P56 ± 3), decapitated them, removed the brains, and transferred them immediately to freshly prepared, ice-cold artificial cerebrospinal fluid (ACSF: 126 mM NaCl, 20 mM NaHCO3, 20 mM dextrose, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 2 mM mgCL2, 50 μM DL-AP5 sodium salt, 20 μM DNQX, and 0.1 μM tetrodotoxin, mixed then bubbled with 95% O2/5% CO2 carbogen gas). We sectioned the brains to generate 400 μm–thick sections using a Leica VT1000S vibratome with a chilled chamber, and immediately transferred the slices to a bubbled ACSF-containing chamberat room temperature. Individual slices of interest were microdissected in a Petri dish while submerged in ACSF under a fluorescence dissecting microscope. Dissected tissue was transferred to a microcentrifuge tube containing ACSF with 1 mg/mL pronase (Sigma, Cat#P6911) for 70 min at room temperature. After incubation, with tissue pieces settled at the bottom of the tubes, ACSF with pronase was exchanged twice with ACSF containing 1% fetal bovine serum (FBS). We next dissociated the tissue into a single-cell suspension by trituration through Pasteur pipettes with polished openings of 600 μm-, 300 μm-, and 150 μm-diameter. 500 cells were sorted into a well of an 8-well PCR strip containing 2 μL ACSF on a BD FACSAriaII SORP with a 130 μm nozzle at 10 psi sheath pressure, and in the single-cell sorting mode. We excluded dead cells by labelling with DAPI (DAPI*2HCl, Life Technologies Cat#D1306) added to the single-cell suspension at 2 ng/mL. We retained only cells that had high tdTomato fluorescence and low DAPI labeling. Immediately after FACS collection, cells were lysed with 25 µL Lysis Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1 % IGEPAL CA-630). Nuclei were pelleted by centrifugation at 500 x g for 30 minutes at 4 °C in a refrigerated microcentrifuge. After pelleting, the supernatant was removed and cells were resuspended by repeated pipetting in 25 µL Reaction Mix (12.5 µL Nextera TD Buffer, 2 µL Nextera TD Enzyme, and 10.5 µL water). Samples were then tagmented by incubation at 37 °C for 1 hour on a heat block. After tagmentation, the reactions were stopped with addition of 5 µL Cleanup Buffer (900 mM NaCl, 300 mM EDTA), 2 µL 5% SDS, and 2 µL Proteinase K and incubation at 40 °C for 30 min. Tagmented DNA was purified using AMPure XP beads at a ratio of 1.8:1 beads to reaction volume, with a final resuspension volume of 11 µL. For indexing and amplification, we added 15 µL KAPA HotStart Ready mix (Kapa Biosystems, Cat# KK2602) and 2 µL each of Nextera i5 and i7 indexed amplification primers. These samples were incubated at 72 °C for 3 minutes, then PCR amplified as follows: 95 °C for 3 min; 9 cycles of 98 °C for 20 sec, 65 °C for 15 sec, and 72 °C for 15 sec; 72 °C final extension for 1 min. Samples were then purified using AMPure XP as above, re-amplified for additional 9 cycles under the same conditions, and then purified once more as before using Ampure XP beads to produce 11 µL final volume. Library quality and quantity were assessed using 1 µL of the final, purified DNA on a BioAnalyzer High Sensitivity DNA chip. For mES cell populations, ES cells grown on mouse embryonic fibroblast (MEF) feeders were passaged onto gelatin plates to dilute/remove feeder cells, and they were made into single-cell suspensions by treatment with Trypsin-EDTA (Thermo Fisher Scientific, Cat#25300054). Cells were washed twice in PBS with 1% FBS, then were resuspended in PBS with 1% FBS and 2 ng/mL DAPI. 500-cell populations of DAPI-negative ES cells were sorted into individual wells of an 8-well PCR strip containing 2 uL of PBS.
attributes.tag: GEO Accession
attributes.value: GSM2333632