GSM8648650: Gut_E5_replicate1; Gallus gallus; RNA-Seq
Dissected frozen gut pieces were resuspended into 600µl of cold homogenization buffer that consisted of 10mM Tris Hcl pH 7.5, 146mM NaCl, 1mM CaCl2, 21mM MgCl2, 0.03% Tween20, 0.01% BSA, 10% EZ buffer (Sigma) and 0.2U/µl Protector RNase Inhibitor (Roche). Tissues were then transferred into 2ml Dounce (Kimble) and homogenized using 8 strokes of the loose pestle followed by 10 strokes of the tight pestle to release nuclei, on ice. Homogenate was then strained through a 40μm cell strainer (Pluriselect) and centrifuged at 500xg for 5 minutes to pellet nuclei. After removing supernatant, nuclei were washed in 1ml resuspension buffer containing 10mM Tris Hcl pH 7.5, 10mM NaCl, 3mM MgCl2, 1% BSA and 0.2U/µl Protector RNase Inhibitor and centrifuged at 500xg for 5 minutes. Nuclei were then washed again in 1ml resuspension buffer followed by centrifugation at 500xg for 5 minutes. Nuclei are then resuspended in resuspension buffer, strained through a 70μm cell strainer (Pluriselect) and finally diluted in resuspension buffer to a concentration of 1200 nuclei/µl before encapsulation in 10x Chromium. All steps were carried on ice or at 4°C. Libraries were prepared according to 10x Genomics 3' version 3.1 chemistry & Illumina protocols