RNA-Seq of HBEC3-KT cells: Mycoplasma pneumoniae-infected day 1 1
HBEC3-KT cells infected with Mycoplasma pneumoniae, as well as uninfected cells, were scraped directly from Transwell membranes into DNA/RNA Shield (Zymo Research). The lysate was transferred to a microcentrifuge tube and stored at -80C. RNA extraction and sequencing were performed at Rush University Genomics Core (Chicago, IL, USA). RNA was purified using the Promega Maxwell RSC simplyRNA Cells Kit (AS1390). RNA quality was evaluated using High Sensitivity RNA ScreenTape Analysis and RNA ScreenTape Analysis (Agilent). Polyadenylated transcripts were enriched with NEXTFLEX Poly(A) Beads 2.0 (PerkinElmer/NovaBio), followed by directional library construction using the NEXTFLEX Rapid Directional RNA-Seq Automation Kit 2.0. Poly(A) selection and library preparation were carried out on the Revvity Sciclone G3 NGSx iQ Workstation. Final libraries were sequenced on an Illumina platform.