Extended Data Fig. 1: Optimization of the experimental sc-SPORT workflow.
From: RNA structure profiling at single-cell resolution reveals new determinants of cell identity
a, Top: NAI-N3 reactivity of the Tetrahymena ribozyme was mapped to its secondary structure. The reactivity was normalized as previously described. Bottom: Boxplot showing the distribution of reactivities for bases in base-paired between adjacent base pairs (left), paired in terminal base pair (middle), and unpaired regions (right) along the Tetrahymena ribozyme RNA. The nucleotide numbers are as shown. The box plots show the mean and 25th to 75th percentile inter-quartile range, the bars show the range from 5th to 95th percentile. b, Gel image showing the distribution of the length of cDNA fragments after 4, 8, and 16 hours of reverse transcription in DMSO and NAI-N3 treated RNAs. The first lane shows the DNA ladder (bases). c, Barplot showing the average fraction of viable cells after treatment with DMSO (grey), NAI-N3 (blue), and DMS (orange) at various concentrations. The center represents mean, and the error bars show the standard deviation. N = 4 biological replicates. d, Line plots showing the distribution of 5 ng hESC total RNA segment lengths before (black) and after fragmentation, by dNTP (orange) and Mg2+ (blue), under different conditions. e, Bioanalyzer plots showing the size distribution of 50 ng hESC total RNA segment lengths before (top) and after fragmentation with 1 mM dNTP (Middle left and Bottom left), or without dNTP (Middle right and Bottom right). f, Density plot of the RNA fragment length distribution treated with either DMSO (red), 25 mM NAI-N3 (blue), or 50 mM NAI-N3 (green) using Agilent Bioanalyzer. g, Barplot showing the amount of DNA generated after library preparation when the starting RNA is unfragmented (left) or fragmented (right). Starting RNA is from 10 cells and is either treated with DMSO (blue), 25 mM NAI-N3 (orange), or 50 mM NAI-N3 (grey). The center represents the mean, and the error bars show the standard deviation. N = 3 biological replicates.
