Journal: bioRxiv

Article Title: Thiol-linked alkylation for the metabolic sequencing of RNA

doi: 10.1101/177642

Figure Lengend Snippet: Thiol-linked alkylation for the metabolic sequencing of RNA (SLAM-seq). (a) Workflow of SLAM-seq. Working time for alkylation and Quant-seq library preparation are indicated. (b) Representative genome browser screen shot for three independent mRNA libraries generated from total RNA of mES cells, prepared using standard mRNA sequencing (top panel), Cap-seq (middle panel) and mRNA 3′ end sequencing (bottom panel; RPM, reads per million,). A representative area in the mouse genome encoding the gene Trim 28 is shown. Bottom shows zoom into 3′ UTR of Trim28. Unnormalized coverage plots of Quant-seq libraries prepared from untreated mES cells or mES cells subjected to s 4 U-metabolic labeling using 100 μM s 4 U for 24 h followed by SLAM-seq. A subset of individual reads underlying the coverage plots are depicted. Asterisks indicate T>C conversions (red) or any conversion other than T>C (black). (c) Conversion rates in 3′ UTR-mapping reads of Quant-seq libraries, prepared from mES cells before (no s 4 U) and after metabolic labeling for 24 h using 100 μM s 4 U (+s 4 U). Dashed line represents expected background sequencing error rate. Median conversion rate across the indicated number of transcripts (n) is indicated. P-value (Mann-Whitney test) is indicated. (d) Relative coverage across transcripts for mRNA-seq and Quant-seq. T>C conversion rate (Conv.) distributes evenly within Quant-seq-covered areas.

Article Snippet: Cap-seq libraries were prepared as previously described . mRNA 3′ end sequencing was performed using the Quant-seq mRNA 3′ end library preparation kit (Lexogen) according to the instructions of the manufacturer.

Techniques: Sequencing, Generated, Labeling, MANN-WHITNEY