spenthil

While I was reading through RNA-seq protocols, I wondered what the rational is behind how protocols fragment cDNA. With some digging I found this nice paper  by Mortazavi, Williams, et al. which gave some reasoning and accompanying investigation.

The rationale for using hydrolysis of RNA before random priming rather than fragmentation of cDNA at the next step was twofold. First, cDNA priming at putatively random sites, if fully successful, will over-represent 5′ ends of transcripts, and this uneven representation will have differing impact on RNAs of different sizes. Second, preliminary data strongly suggested that there are some strongly favored and disfavored sites of random priming, and we observed this in samples that were primed without hydrolysis. It did not, however, correspond to simple GC content bias (Supplementary Fig. 1b). We reasoned that, at room temperature, some RNA secondary structure may shield parts of transcripts from priming while favoring other sites. By fragmenting the RNA, we expected to reduce the amount of such secondary structure, though not completely eliminate it. RNA fragmentation before copying would also be expected to greatly reduce 5′ bias. This protocol gave better overall uniformity than protocols without RNA fragmentation (Supplementary Fig. 1), although some residual and reproducible nonuniformity clearly persists for randomly primed substrates that was not observed in other kinds of Illumina sequencing substrates handled simultaneously, such as chromatin immunoprecipitation sequencing (ChIPSeq) samples (for example, Supplementary Fig. 1c).

The following are some things I have learned to be quicker with terminals while keeping the damage to my hands at a minimum. The tips focus around BASH and Vim keyboard shortcuts, but should have analogs in other environments. Read the rest of this entry »