GSM4726540: Mm_CD4_acute_I; Mus musculus; RNA-Seq
At the designated chronic or acutetimepoint mice received a lethal dose of a Ketamine-Xylazine mixture (Ketamine320 mg/kg; Xylazine 5 mg/kg) i.p. and were transcardially perfused with 50ml ofice cold PBS. spinal cord tissue was removed and placed in a small petri dishand fully covered with ice cold D-PBS (with Ca2+, Mg2+,glucose and pyruvate, Gibco). Now under sterile conditions, tissues werechopped into smaller pieces and transferred into C-tubes® containing enzyme mix 1 and 2 (for furtherdetails please refer to manufactures protocol). The tissue was homogenized onthe gentleMACS® Octo Dissociator with Heaters (Miltenyi), program 37C_ABDK_01. Followinghomogenization, C-tubes® containing homogenate were briefly centrifuged, resuspendedin 10 ml of cold D-PBS then passed through a 100 µm cell strainer placed on anew 50 ml tube. The cell suspension was centrifuged at 300 x g and 4 °C for[TC1] 10 min. The supernatant was completely aspirated and the cell pelletwas at first resuspended with 3.1 ml cold D-PBS and 900 µl Percoll®, transferredinto a new 15 ml tube and then overlaid with 4 ml cold D-PBS. To remove most ofthe cell debris and myelin a density gradient centrifugation step was performedat 3,000 x g and 4 °C for 10 min with a soft acceleration and brake. The twotop phases were aspirated, the 15 ml tube was filled with cold D-PBS, gentlyinverted, centrifuged at 1,000 x g and 4 °C for 10 min with full accelerationand brake. Again, the supernatant was aspirated and now an erythrolysis wasperformed by adding 10 ml of 1X Red Blood Cell Removal Solution® followed by anincubation of 10 min at 4 °C. The lysis was stopped by adding 10 ml of coldD-PBS/BSA buffer (0.5 % BSA, pH 7.2). A final washing step at 300 x g and 4 °Cfor 10 min prepared the single cell suspension for the following immunostainingprocedure. Libraries were prepared as per manufacturer's insturctions provided by 10X Genomics platform for the Single Cell 3-prime Gene Expression v3 workflow. scRNA seq