title: GSM9204065: Pooled SHARE-seq libraries, batch 2 containing sub-libraries 3.3, 3.4, 4.3, 4.4; Mus musculus; OTHER
library_construction_protocol: SHARE-seq was performed on V1 tissue harvested from P7, P14, P21, P28, and P35 C57BL/6 mice raised in DR or NR conditions (n = 4 mice per condition and time point). Mice were deeply anesthetized with isoflurane, and V1 was dissected. The tissue was dounce homogenized in 1.5 mL of cold homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM tricine-KOH pH 7.8, 1X EDTA-free protease inhibitor cocktail (PIC) tablet (Sigma Aldrich 11873580001), 1 mM dithiothreitol (DTT), 0.15 mM spermine, 0.5 mM spermidine, 0.1 U/μL Enzymatics RNase inhibitor (Y9240L), 0.05 U/μL SUPERase inhibitor (AM2696)) with a tight pestle for 20 strokes. The sample was then supplemented with 0.3% IGEPAL CA-630 and further dounced 5 strokes with a tight pestle. Homogenate was filtered through a 40-μm strainer into a 15 mL conical tube, and 3.5 mL of homogenization buffer and 5 mL of 50% OptiPrep solution (50% OptiPrep (Sigma D1556), 25 mM KCl, 5 mM MgCl2, 20 mM Tricine-KOH pH 7.8, 1X PIC, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, 0.1 U/μL Enzymatics RNase inhibitor, 0.05 U/μL SUPERase inhibitor) were added. Tissue lysate was underlaid with 1 mL of 30% OptiPrep solution and 1 mL of 40% OptiPrep solutions. Samples were then centrifuged at 10,000g for 18 min at 4°C in an ultracentrifuge. Following centrifugation, approximately 250 μL of nuclei were collected at the 30/40% OptiPrep interface and diluted in NIB-RI buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630, 0.1 U/μL Enzymatics RNase inhibitor, 0.05 U/μL SUPERase inhibitor). The nuclei were then centrifuged at 500g for 10 min at 4°C in a swinging-bucket centrifuge and subsequently resuspended in NIB-RI buffer containing 0.2% formaldehyde. Samples were incubated at room temperature for 5 min and then quenched on ice for 5 min with the addition of 125 mM glycine, 40 mM Tris pH 8, and 0.08% BSA. Nuclei were then washed two times in NIB-RI buffer by centrifuging at 500g for 10 min at 4°C and replacing the supernatant without disturbing the pellet. After the second wash, the supernatant was removed. Nuclei were flash-frozen in liquid nitrogen and then stored at -80°C until all samples were collected.