Loss of Zfp335 triggers cGAS/STING-dependent apoptosis of post-B-selection pre-T cells [TCRa-seq]

The transcription factor Zfp335 is esstential for the survival of post-B-selection DN4 thymocytes. We utilized TCRa repertoire sequencing to assess alterations to survival duration for Zfp335-deficient DP thymocytes. Overall design: DP thymocytes were FACS-sorted (Live Lin- CD4+ CD8+) from Zfp335-fl/fl or Zfp335-fl/fl E8III-cre positive 7 week old male and female mice. Each sample was from one individual mouse. Following sorting, total RNA was isolated and libraries were prepared as follows: 5ng total RNA was reverse transcribed with Trac-specific reverse transcription primer and template switch oligo containing a Unique Molecular Identifier (UMI) using SmartScribe RT (Takara Bio). cDNA was purified and TCRa transcripts were amplified with a TSO-specific nested Trac-specific primers using Q5 polymerase (NEB). Each sample was dual indexed then all samples pooled and Illumina-compatible adaptors were added using the NEBNext Ultra II DNA library prep kit. 300x300bp sequencing was performed on an Illumina MiSeq instrument.