A Small RNA isolation and sequencing protocol and its application to assay CRISPR RNA biogenesis

Next generation high-throughput sequencing has enabled sensitive and unambiguous analysis of RNA populations in cells. Here, Stanford University researchers describe a method for isolation and strand-specific sequencing of small RNA pools from bacteria that can be multiplexed to accommodate multiple biological samples in a single experiment. Small RNAs are isolated by polyacrylamide gel electrophoresis and treated with T4 polynucleotide kinase. This allows for 3′ adapter ligation to CRISPR RNAs, which don’t have pre-existing 3′-OH ends. Pre-adenylated adapters are then ligated using T4 RNA ligase 1 in the absence of ATP and with a high concentration of polyethylene glycol (PEG). The 3′ capture step enables precise determination of the 3′ ends of diverse RNA molecules. Additionally, a random hexamer in the ligated adapter helps control for potential downstream amplification bias. Following reverse-transcription, the cDNA product is circularized and libraries are prepared by PCR. The researchers show that the amplified library need not be visible by gel electrophoresis for efficient sequencing of the desired product. Using this method, they routinely prepare RNA sequencing libraries from minute amounts of purified small RNA. This protocol is tailored to assay for CRISPR RNA biogenesis in bacteria through sequencing of mature CRISPR RNAs, but can be used to sequence diverse classes of small RNAs. The researchers also provide a fully worked example of their data processing pipeline, with instructions for running the provided scripts.

rna-seq The flowchart of the major steps in the protocol

Silas S, Jain N, Stadler M, Fu BXH, Sánchez-Amat A, Fire AZ, Arribere J. (2018) A Small RNA Isolation and Sequencing Protocol and Its Application to Assay CRISPR RNA Biogenesis in Bacteria. Bio Protoc 8(4). [article]

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