Fig. 1. Design and optimization of MSR-seq.

a Schematic representation of multiplex small RNA-seq. The required steps are indicated in solid arrows and optional steps in dashed arrows. Symbols are explained in the box. b Features of the capture hairpin oligo (CHO) with detailed descriptions for each feature. c Fraction of ligation products for the test RNA oligonucleotides containing rA, rC, rG, and rU at the 3′ end. n = 3 independent experiments. Quantitative data were from denaturing gels stained with SYBR Gold shown in Supplementary Fig. 1a. The mean value is shown as a bar; the mean of all replicates is shown as a dashed line. The molar ratio as measured by UV absorbance of input RNA and the CHO in the ligation reaction was ~1:2. Fraction product is calculated as (Product)/(Product + Free CHO). The expected stoichiometric amount of ligation product corresponds to ~0.5. d Fraction of ligation products for the test 5′ biotinylated deoxyoligonucleotides containing dA, dC, dG, and T at the 3′ end. n = 3 independent experiments. Quantitative data were from denaturing gels stained with SYBR Gold shown in Supplementary Fig. 1b. The mean value is shown as a bar; the mean of all replicates is shown as a dashed line. Fraction product is calculated as (Product)/(Product + Free 5′biotin-oligo). e Relative reverse transcription products with RT performed free in solution (off-bead), on streptavidin bead (on-bead), and on-bead without calf-intestine alkaline phosphatase treatment (No-CIP). Input samples were total RNA from HEK293T cells. n = 3 independent experiments. Quantitative data were from native gels stained with SYBR Gold shown in Supplementary Fig. 1c. The mean value of replicates is shown as a bar. Product is defined as all bands above the terminal transferase product of the RT (TdT) band in Supplementary Fig. 1c. Fraction product is calculated as (Product in each sample)/(sample with the maximum amount of product). The non-zero value from No-CIP samples was from spurious oligos associated with the beads, see Supplementary Fig. 1c. f Fraction of reverse transcriptase m¹A58 read-through product with no demethylase treatment/short RT reaction (none), demethylase treatment/short RT reaction (DM), and no demethylase treatment/long RT reaction (overnight RT). Input samples were total RNA from HEK293T cells. n = 3–4 independent experiments. Quantitative data were from native gels stained with SYBR Gold shown in Supplementary Fig. 1e, h. The mean value of replicates is shown as a bar. Product is defined as all bands above the m¹A58 band in Supplementary Fig. 1e, h. Product is defined as all bands above the TdT band in Supplementary Fig. 1c. Fraction product is calculated as (Product)/(Product + m¹A58 stopband).