GSM8919258: D50; Ovis aries; RNA-Seq
This protocol describes the isolation of frozen tissue nuclei, beginning with pre-cooled reagent preparation including lysis buffer (0.25M sucrose, 5 mM MgAc2, 10 mM Tris-HCl pH 8.0, 1 mM DTT, 0.1 mM EDTA, 1x protease inhibitor, 1U/μl RiboLock RNase Inhibitor), iodixanol solution (0.16M sucrose, 10 mM NaCl₂, 3 mM MgCl₂, 10 mM Tris-HCl pH 7.4, 1U/μl RiboLock RNase Inhibitor, 1mM DTT, 0.1mM PMSF protease inhibitor with adjustable iodixanol concentrations at 30%, 33%, or 50%), and nuclear wash buffer (0.04% BSA, 0.2U/μl RiboLock RNase Inhibitor, 500mM mannitol, 0.1mM PMSF protease inhibitor in PBS). Tissue samples require soybean-sized animal tissues (>0.2g) or plant tissues (>1g) that are blotted dry, minced into 1-2mm³ pieces on ice, flash-frozen in liquid nitrogen for 5 minutes, and stored in ultra-low temperature conditions, with an intact quality control sample separately preserved for RNA integrity verification prior to experiments. The nuclear isolation process involves thawing tissue in lysis buffer within a Dounce homogenizer, sequential grinding with loose and tight pestles (15-20 and 5-10 strokes respectively), filtering through a 70μm strainer, mixing filtrate with 50% iodixanol, followed by layered centrifugation using 33% and 30% iodixanol solutions in 15mL tubes to isolate nuclei through density gradient separation. ibrary was performed accordina to the manufacter's instructions (sinale cell 3' y2orotocol10xGenomics).Brieflyresuspended in the master mix andI beads into the chip to generate the geoaceo touetnerwitn nanttionInoead_in-emulsion (GEM) The Dolv-A RNAfrom the cell lysate contained in eveny;inale GEM wasretrotranscripted to cDNAan llwmina Ri orme!WMcncontalneseauence, Uniaue Molecular ldentifer (UMl) and the 10x Barcode. The pooledarcoded cDNA was then cleaned uo with Silane DynaBeads, amplified by PCRand the apropiated sized fragments were selected with SPRlselect reagent forsubseauent library construction. During the library construction llumina R2 prime!seauence, paired-end constructs with P5 and P7 seauences and a sample indexwere added