GSM8281651: Neonate_Thymus_Double_Negative_RNA_2; Mus musculus; RNA-Seq
Thymi and spleens from adult or neonatal mice were collected in RPMI+10% FBS and mashed through a 40µM cell strainer to create a single-cell suspension. Spleens and thymi underwent magnetic enrichment with CD8a and CD4 MicroBeads (Miltenyi Biotec; cat# 130-117-044; cat# 130-117-043), respectively, per the manufacturer's instructions. The bound thymic fraction was sorted for double positive thymocytes, while the eluted fraction underwent CD8a magnetic enrichment as described previously, after which the bound fraction was sorted for SP8 thymocytes, while the eluted fraction was sorted for DN thymocytes. Thymocyte counts were taken by diluting a single-cell thymus suspension and counting on a MoxiZ cell counter. To extract RNA, sorted cells were resuspended in 1ml of Trizol (Thermo #15596018). 200ul of chloroform was added to 1ml of trizol lysates. After vigorous shaking, samples were centrifuged at 16000x g at 4C. During the spin, phase lock gel tubes were prepared by a pre-spin 12000xg at room temperature. 600ul chloroform was added to these pre-spun tubes. The aqueous phase of trizol-chloroform mixture was added to the phase lock tube containing chloroform. After vigorously shaking these phase lock tubes were centrifuged at 12000xg at 4 C. The aqueous phase from the phase lock tubes was added to isopropanol and glycoblue containing precipitation tubes. These precipitation tubes were mixed, incubated for 1 hour, and spun at 16,000 x g at 4o c. The supernatant was discarded and the pellets washed with ice-cold ethanol. Pellets were further centrifuged at 16000xg at 4 c. The supernatant was discarded, and the pellet air-dried and resuspended in RNAase-free water. RNA sample quality was confirmed using a Qubit (RNA HS kit; Thermo Fisher) to determine the concentration and with a Fragment Analyzer (Agilent) to determine RNA integrity. PolyA+ RNA was isolated with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). TruSeq-barcoded RNAseq libraries was generated with the NEBNext Ultra II Non-Directional RNA Library Prep Kit (New England Biolabs). Each library was quantified with a Qubit (dsDNA HS kit; Thermo Fisher) and the size distribution was determined with a Fragment Analyzer (Agilent) prior to pooling.