GSM4744778: TAZ-CAMTA1iECs; Mus musculus; RNA-Seq
Pooled lungs were isolated from three five week-old TRE-H2B;TRE-TAZ-CAMTA1;Cdh5-tTA that had been induced (switched to normal water from 1.5mg/mL doxycycline water) at birth. Lungs were digested in .2% collagenase 1 (Thermo Fisher) and treated with ACK Lysis Buffer before being mixed with the viability stain 7-AAD (BioLegend) and subjected to flow-activated cell sorting (FACS) at the Moody Foundation Flow Cytometry Core on a FACS ARIA II SORP using FACSDiva Software. Approximately 400,000 cells were obtained, and a viability of 80% was estimated with a Countess Automated Cell Counter (Thermo Scientific). Samples were processed using the 10X genomics platform by the UTSW Next Generation Sequencing Core. The appropriate volume of cells was loaded with Single Cell 3' Gel Beads into a Next GEM Chip G and run on the Chromium Controller. GEM emulsions were incubated and then broken. Silane magnetic beads were used to clean up the GEM reaction mixture. Read one primer sequence was added during incubation and full-length, barcoded cDNA was then amplified by PCR after cleanup. Sample size is checked on the Agilent Tapestation 4200 using the DNAHS 5000 tape. Concentration was determined by the Qubit 4.0 Fluorimeter (Thermo Scientific) using the DNA HS assay. Samples were enzymatically fragmented and underwent size selection before library construction.  During library preparation, Read 2 primer sequence, sample index and both Illumina adapter sequences were added.  Subsequently, samples were cleaned up using Ampure XP beads and post library preparation quality control was performed using the DNA 1000 tape on the Agilent Tapestation 4200. Final concentration is ascertained using the Qubit DNA HS assay. Samples were loaded at 1.6 pM and run on the Illumina NextSeq500 High Output Flowcell using V2.5 chemistry.  Run configuration is 28x98x8.