Fig. 5: The LOI mutant HP results in a higher number of toxic sCAGs in the RISC of transfected cells than the Ref HP.

A Top, the four sequences that were converted to libraries and sequenced by small RNA seq; bottom, schematics explaining two different sequencing approaches. For set 1 two libraries were prepared, one containing a 1:1 mixture of 10 pmol of size markers (19 and 35 nts long) and another containing a 1:1 mixture of 10 pmol of two CAG repeat containing RNAs (21 and 36 nt) and then together subjected to smRNA seq on an lllumina HiSeq 3000. For set 2 the four oligonucleotides were mixed 1:1:1:1 and the resulting library was subjected to sequencing. B Raw counts of the two libraries. Percent of reads that had the exact length and sequence of the RNA that was used are indicated. C Normalized read counts detected bound to the RISC in cells transfected with either 2.5 nM siNT1, (CAG)₂₁, the Ref HP, or the LOI HP after 24 h (all as shown in C). Reads that contain only CAG TNR, CCG TNRs or other sequences are shown. Shown are the averages and the variance between the duplicate samples. D Sequences (top) and length distribution (bottom) of HP derived reads detected in the RISC of transfected cells in D. Solid and stippled lines of the same color represent the two duplicates.