he barcode poly(A) RNAs used to generate the standards, each RNA was 33P labelled at its 5 terminus and analysed on denaturing polyacrylamide gels under conditions that enabled single nucleotide resolution. The values to the right of each panel indicate the modes and approximate ranges of the poly(A) tail lengths (after accounting for the 10 nucleotide barcodes). Also shown are marker lanes with 33P labelled Century Plus ladder (C, Ambion), 33P labelled Decade ladder (D, Ambion) and a partial base hydrolysis ladder of the labelled barcode poly(A) RNA used to make the 324 nucleotide standard of mix 2 (OH). c, The relative PAL seq yield of each poly(A) length standard. Saucony Pink And Brown
composition of positions 23 in either uniquely genome or standard mapping tags that extended the full length of the reads (36 nucleotides).
Box plots show the distribution of yields for 32 PAL seq libraries (line, median; box, 25th and 75th percentiles; whiskers, 10th and 90th percentiles). a, Evidence against non specific RNA degradation. Plotted are nucleotide identities at the positions immediately Saucony upstream of poly(A) tags that both mapped uniquely to the genome (or standards) and ranged from 22 nucleotides in length (a range chosen to be long enough to enable mostly unique mapping to the genome, yet short enough to include enough 5 adaptor nucleotides in a 36 nucleotide read to clearly identify the 5 end of the tag). Frequencies were normalized to the aggregate nucleotide Saucony Boston Public Gardens
standard is plotted, after normalizing to the starting ratio determined from analysis of 5 mix on a denaturing polyacrylamide gel.
For each standard in the indicated mix, the yield of poly(A) tags relative to that of the A10 Saucony New York Manhattan
Because RNase T1 cuts after Gs, the nucleotide preceding each 22 tag was expected to be G, unless the mRNA had been cut for some other reason. The high frequency of G indicated that most mRNA fragments had not been cut for other reasons, which also implied that for these samples the poly(A) tails had also remained intact. We are unable to explain the high .
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