Extended Data Fig. 1: ARTR-seq setup and condition optimization.
From: Profiling of RNA-binding protein binding sites by in situ reverse transcription-based sequencing
a, Functional domains of the engineered MMLV RTase (H8Y, D200N, T306K, W313F, T330P, D524G, L603W)24,25. The MMLV RTase (full length) is composed of three domains: polymerase (red), connection (purple) and RNase H (yellow). The RNase H domain and the first 24 N-terminal residues were omitted in MMLV RTase (25-497) to improve its RT activity71. b, Coomassie bright blue staining of three purified pAG-RTase fusion constructs with a link length of 30 amino acids. c, qRT-PCR analysis for ACTB, METTL14 and RBM15 showing the relative RT activity of three tested purified pAG-RTase fusion proteins. Two commercial RTases SuperScript II and SuperScript III were loaded as positive controls. n = 3 biological replicates. d, qRT-PCR showing the relative RT efficiency using the indicated random primers. pAG-MMLV RTase fusion protein (25-497) was used in this analysis. n = 2 biological replicates. e, qRT-PCR showing the effects of different biotinylated dNTPs on the relative RT efficiency using pAG-MMLV RTase (25-497). Biotin-16-dUTP and biotin-16-dCTP exhibited the least hindrance to RT efficiency. Both were used in the ARTR-seq procedure by mixing with regular dTTP and dCTP at a 1:1 ratio. n = 1 biological replicate. f, Immunofluorescence (IF) imaging of the secondary antibody (2nd Ab, yellow), pAG-RTase (red), newly synthesized cDNA (green), and nucleus (blue) for RBFOX2 ARTR-seq. Scale bars, 10 μm. g, qPCR analysis to measure relative cDNA yields of ARTR-seq samples. n = 2 biological replicates.
