Fig. 1

Workflow of polysome profiling and sample selection for bisulfite (bs)RNA-seq. HeLa cell lysates were separated by ultracentrifugation through linear sucrose density gradients. Twenty-four fractions per gradient were taken and combined for subsequent analyses as indicated. The four pooled fractions chosen for bsRNA-seq are indicated by blue boxes. a Principle of using sucrose density gradient ultracentrifugation to separate mRNAs by ribosome association (top) and scheme for merging the 24 fractions into different pools for downstream analyses (bottom). First, subsamples were taken and three adjacent fractions were merged to generate eight samples for Western blotting (Protein Fractions). Second, fractions were spiked with a Renilla luciferase (R-Luc) in vitro transcript and combined pairwise to generate 12 merged fractions (RNA Fractions). Total RNA was isolated, DNase treated, assessed for integrity and used for RT-qPCR. Third, bsRNA-seq Fraction pools were created as follows: (pool 1: RNA Fractions 2–3; pool 2: 4–5; pool 3: 6–8; pool 4: 9–11). 10 μg of total RNA from each pool was spiked with the ERCC in vitro transcripts, rRNA depleted and sodium bisulfite treated prior to library construction and high-throughput Illumina sequencing. b Distribution of Ribosomal Protein L26 (RPL26) across gradients. Protein fractions were subjected to western blotting (equal proportions were loaded). Replicate B is shown as an exemplar of all biological replicates. c Absorbance traces (254 nm) across the three biological replicate gradients processed for bsRNA-seq. d Distribution of tRNA and rRNA across gradients. RNA fractions were analysed by microfluidic electrophoresis (equal proportions were loaded). The pseudo-gel image for replicate E is shown (see Fig. S1A for data from all replicates). e Distribution of representative mRNAs across gradients were determined by RT-qPCR. Results for three mRNAs of different coding region length are shown: RPS3 (ribosomal protein S3), CCND1 (cyclin D1) and SZRD1 (SUZ RNA binding domain containing 1) (see Fig. S1B for further examples). mRNA levels were normalised to R-Luc, rescaled as percentage of total signal, and are shown as mean with error bars indicating ± standard deviation