DRP004815
For high throughput profiling of nascent nuclear transcripts in N4bp1 deficient inflammatory responses , metabolically 5EU labelled RNAs from N4bp1 KO macrophages stimulated with Pam3CSK4 (TLR2 ligands) were isolated and subjected to RNA sequencing.
Thioglycollate induced peritoneal macrophages from wild-type or N4bp1 KO mice were stimulated with Pam3CSK4 for indicated time (0.6 to 3.6h), then nascent nuclear transcripts were pulsed by adding 5EU (5-ethyl uridine) in culture medium for additional 20 to 30min. 5EU labelled RNAs were isolated with Click-iT Nascent RNA Capture Kit (invitrogen) following manufacture's protocol, and subujected to reverse transcription with ReverTra Ace qPCR RT Master Mix (TOYOBO).