Extended Data Fig. 1: Optimization of RT&Tag.
From: Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation
a) Performance comparison of RT&Tag using biotinylated or un-biotinylated oligo(dT)-adapter B fusion oligonucleotides based on the following metrics: roX2 enrichment for MSL2 (left) and number of differentially enriched transcripts for K27me3 (right) based on 2 replicates. Both experiments were performed using reverse transcription performed at the same time as tagmentation (CoTagRT) approach. b) Performance comparison of RT&Tag if reverse transcription is performed prior to addition of pA-Tn5 (preTagRT) or if reverse transcription is performed at the same time as tagmentation (CoTagRT). Both experiments were performed using un-biotinylated oligo(dT)-adapter B fusion oligonucleotides. Performance of RT&Tag was assessed based on the following metrics: roX2 enrichment for MSL2 (top left), number of differentially enriched transcripts for K27me3 (top right) and number of differentially enriched transcripts for m6A (bottom) with pre-TagRT versus Co-TagRT based on 2 replicates. Differential enrichment was defined as >2-fold change for K27me3 or >1.5-fold change for m6A, <0.05 FDR. c) Density plots showing the distribution of aligned MSL2 (top) and H3K27me3 (bottom) RT&Tag reads (n = 2) scaled over Drosophila gene bodies for biotinylated oligo(dT) CoTagRT (left), unbiotinylated oligo(dT) CoTagRT (center), and unbiotinylated oligo(dT) preTagRT (right) RT&Tag variations. A clear bias towards the 3’ end of genes is observed under all conditions.
