How RNA sequencing is done?

RNA sequencing (RNA-seq) is a powerful technique used to analyze the transcriptome of a sample, which refers to all the RNA molecules present at a specific time. Here's a more detailed overview of how RNA sequencing is typically done:

·         Sample Collection and RNA Extraction: RNA is extracted from the cells, tissues, or organisms of interest. This can involve various methods depending on the sample type and downstream applications. The extracted RNA should ideally represent the full spectrum of RNA molecules present, including messenger RNA (mRNA), non-coding RNA (such as long non-coding RNA and microRNA), and ribosomal RNA (rRNA).

·         RNA Quality Control: The quality and integrity of the extracted RNA are assessed using techniques like spectrophotometry (measuring absorbance at specific wavelengths) and gel electrophoresis. High-quality RNA with minimal degradation is crucial for accurate sequencing results.

·         Library Preparation: The extracted RNA undergoes library preparation, where the RNA molecules are converted into a library of cDNA fragments suitable for sequencing. This process typically involves the following steps:

·         RNA Fragmentation: The RNA molecules are fragmented into smaller pieces. Fragmentation can be achieved chemically or enzymatically.

·         Reverse Transcription: The fragmented RNA is reverse transcribed into complementary DNA (cDNA) using reverse transcriptase enzymes. This step converts RNA into a more stable and amplifiable form for sequencing.

·         Adaptor Ligation: Sequencing adapters containing sequences necessary for sequencing (such as primer binding sites) are ligated to the ends of the cDNA fragments. Adapters may also contain unique molecular identifiers (UMIs) or barcodes to enable multiplexing, allowing multiple samples to be sequenced together.

·         PCR Amplification: The cDNA fragments with ligated adapters are amplified via polymerase chain reaction (PCR) to generate enough material for sequencing.

·         Sequencing: The prepared RNA-seq libraries are then sequenced using high-throughput sequencing platforms such as Illumina sequencing systems. During sequencing, fluorescently labeled nucleotides are added to the growing DNA strands, and the emitted light signals are detected and recorded to determine the sequence of each fragment.

·         Data Analysis: After sequencing, the raw data (sequencing reads) are processed and analyzed bioinformatically. This typically involves the following steps:

·         Quality Control: Assessing the quality of sequencing reads and removing low-quality reads and sequencing artifacts.

·         Alignment: Mapping the sequencing reads to a reference genome or transcriptome to determine where each read originated.

·         Quantification: Estimating the abundance of RNA transcripts by counting the number of reads that map to each gene or transcript.

·         Differential Expression Analysis: Comparing gene expression levels between different conditions or experimental groups to identify genes that are differentially expressed.

·         Functional Analysis: Interpreting the biological significance of the RNA-seq results, such as identifying enriched biological pathways or functional categories.

RNA sequencing enables a wide range of applications, including gene expression analysis, transcriptome profiling, alternative splicing detection, identification of non-coding RNAs, and exploration of RNA modifications and dynamics.