GSM8164495: NL replicate1; Mus musculus; RNA-Seq
Mouse embryos were flash frozen in liquid nitrogen and minced to smaller chunks on ice with sterile micro-dissection scissors. The finely minced tissue were transferred to a pre-chilled 2 mL Dounce homogenizer containing 2 mL cold 1x Homogenization Buffer (HB) buffer which contained 0.26 M Sucrose, 0.03 M KCl, 0.01M Mg2Cl, 0.02M Tricine-KOH pH 7.8, and Ultra-Pure Water (Thermo-Fisher), with the following added on the day of tissue processing— 1mM DTT, 0.5 mM Spermidine (Sigma Aldrich), 0.15mM Spermine (Sigma Aldrich), 0.3% NP40, 1x Roche Complete Protease Inhibitor Cocktail, 1U/μl RNase Inhibitor (Fisher Scientific). The tissue was homogenized on ice by moving a 'loose' pestle up and down 10-15 times to disrupt tissue structure, followed with a 'tight' pestle to achieve a final homogenate. This homogenate was then strained through a 70μm Flowmi strainer into a 2 mL DNA/RNA LoBind tube and centrifuged at 4°C at 350 rcf for 5 minutes. After the centrifugation, the supernatant was discarded, and the pellet was washed with 1 mL cold PBS, passed through a 40μm Flowmi strainer, and centrifuged again. For density gradient separation, the isolated nuclei were resuspended in 400μl of 1x HB and combined with an equal volume of 50% chilled Iodixanol solution. Subsequent layers of 30% and 40% Iodixanol solutions, diluted in 1x HB, were gently layered under the initial mixture to create a gradient. In the final step, the tube with the nuclei was centrifuged using a pre-cooled bucket-rotor centrifuge at 3,000 rcf for 20 minutes at 4°C. After centrifugation, the top layer was carefully removed, without disrupting the “fuzzy” white ring of nuclei. A narrow-bore pipette tip was used to collect no more than 200μL of the nuclei, which were then mixed with an equal volume of 1x HB. Nuclei concentration was determined using a Countess II FL automated cell counter (Thermo Fisher Scientific), adjusting to a final concentration optimal for 10x Genomics Chromium library preparation. Transposed nuclei are combined with GEL beads that contain barcodes and partiotioned on a Chromium Next GEM chip to form Gel Beads in emulsion (GEMs). The Gel Bead contains an Illumina TruSeq Read 1 primer, 16 nt 10x Barcode, 12 nt UMI, and a 30 nt poly-dT sequence which are released upon GEM formation. Incubation of the GEMs leads to barcoded cDNA production from the mRNA to create the gene expression library.