RNA sequencing Overview

 We started the week inoculating 3 plates and 3 flasks from freeze back, then later in the week did a pre-desiccation procedure and discussed more about what the RNA sequencing would look like. 

For pre desiccation we did the usual procedure, let it dehydrate for two days then placed it in the Egg, which is our new desiccator. Unlike the old glass one, this one is sealed and vacuumed making it true desiccation. Dehydration is a process of removing water or moisture from a substance, typically through heat, air circulation, or other drying methods. In dehydration, some water remains within the material, and the substance typically retains some degree of moisture. True desiccation, on the other hand, is a more extreme form of water removal that aims to completely eliminate all moisture from a substance. 

A vacuum is not absolutely necessary for true desiccation, but it can be an extremely effective method for removing moisture. In the desiccation process, a vacuum proves valuable by comprehensively addressing moisture removal through several key mechanisms. By lowering atmospheric pressure, a vacuum reduces the boiling point of water, which means moisture can be extracted more easily and at lower temperatures. This creates optimal conditions for facilitating more complete moisture removal, essentially allowing water molecules to transition to vapor more readily. Additionally, the vacuum environment prevents moisture reabsorption from the surrounding atmosphere, ensuring a more thorough and stable desiccation process.


Pre-desiccation cell packing 

1. Normalize 3ml of culture to an OD between 0.95-1.00

2. Spin down 1ml of media, remove supernatant and resuspend pellet in nuclease free water

3. Resuspend pellet, then centrifuge and remove supernatant

4. Add the second ml of culture, repeat washing steps

5. Add the third ml of culture, repeat washing steps

6. Plate 100ul dots into 1 inch kapton squares in a 6 well plate in triplicate



The process of RNA sequencing begins with sample preparation and careful RNA isolation, ensuring high-quality RNA extraction while minimizing degradation. Subsequent purification focuses on isolating messenger RNA through methods like poly-A selection or rRNA depletion. Library preparation transforms RNA into sequencing-compatible fragments by fragmenting RNA, synthesizing complementary DNA (cDNA), adding sequencing adapters, and performing PCR amplification. High-throughput sequencing platforms generate millions of short DNA sequence reads, which are then subjected to rigorous computational analysis. This computational stage involves quality control of sequencing reads, alignment to reference genomes or transcriptomes, read count quantification, data normalization, and statistical analysis to identify differentially expressed genes. The final stage encompasses data interpretation, including biological pathway analysis, gene ontology enrichment, functional annotation, and validation of key findings through alternative methodological approaches.

Library Preparation Once high-quality RNA is isolated, the next step is library preparation, which transforms RNA into sequencing-compatible DNA fragments. This involves several key processes:

  • Reverse transcription of RNA to complementary DNA (cDNA)
  • Adding sequencing adapters to the cDNA fragments
  • Selecting specific RNA types (often mRNA via poly-A selection or rRNA depletion)
  • Fragmenting the cDNA to create a suitable size range for sequencing
  • Amplifying the library through PCR to generate sufficient material

Sequencing Modern RNA sequencing typically employs high-throughput platforms like Illumina's next-generation sequencing technologies. These platforms generate millions of short DNA sequence reads that will later be computationally mapped back to a reference genome or transcriptome. The depth of sequencing (number of reads) is crucial - deeper sequencing provides more comprehensive coverage and increased sensitivity for detecting low-abundance transcripts.

Bioinformatics Analysis The computational analysis of RNA sequencing data is complex and involves multiple sophisticated steps:

  1. Quality filtering of sequencing reads
  2. Alignment of reads to a reference genome or transcriptome
  3. Quantification of gene expression levels
  4. Normalization to account for technical variations
  5. Statistical analysis to identify differentially expressed genes

Comments

Post a Comment

Popular posts from this blog

Summary of 'the effect of magnetic field on the activity of superoxide dismutase'

  Magnetic fields are created when electrons spin. Normally electrons are paired, and if they spin in opposite directions from magnetic fields cancel out. However in free radicals the electron sometimes are unpaired or a pair electrons spin in the same direction, in this case the magnetic fields are not cancelled out. Most enzymes are not affected by these magnetic fields, however some are. This experiment is to determine the amount of effect of a magnetic field on the enzyme known as superoxide dismutase aka SOD.   SOD is a catalyst that catalyzes the conversion of superoxide free radicals into O2 and H2O2. This experiment was to study the influence of magnetic fields on SOD activity, to see how it would affect biological processes. They tested this by exposing SOD to EMF for various amounts of time then measuring its activity by measuring its reaction to oxygen. To conduct the experiment, the SOD enzyme was combined with Na2CO3, methionine, NBT, EDTA, PBS, dH20 and riboflavin Sol
Read more

Aquaticus Lyse

Image
      In the last blog I mentioned moving onto another project with Kieran, trying to replicate a D.rad lyse onto aquaticus. Before I could start with the procedures I had to make the solutions and create the protocols. It was a lot of dilutions and changing the pH of things, but I also had to make an EDTA solution.       To make the multibuffer I would need, I started by making 100 ml of 50 mM sucrose, and 100 ml of 10 mM tris-HCL. After making those two solutions, I made 100 ml of .5 M, pH 8.0 EDTA. While it was simple enough to combine the ingredients (18.61 g disodium EDTA- 2 H2O and 80 ml water), I then had to change the pH. The original pH was around 3.4, so it took a while, dosing it with HCl to raise the pH and cause the solids to dissolv e. After the EDTA was diluting the 10% Triton x-100 to .1%, which was done when creating the multibuffer. I ended up making 100 ml of multibuffer, with 8 ml of the EDTA solutiom, 5 ml of the sucrose, 1 ml of the 10x Triton x-100, 1 ml of the
Read more

Linear Plasmids, Restriction Enzyme Ligation and Plasmid Extractions

Image
  Linear Plasmids, Restriction Enzyme Ligation, and Plasmid Extractions   The project I’m currently working on is linear plasmids. We’re trying to take a plasmid from E.coli that contains a gene called tet that codes for tetracycline (an antibiotic) resistance, and remove D.rad’s (Deinococcus Radiodurans) lux.s gene before incorporating tet into the spot in D.rads genes where lux used to be.   On Monday we decided to separate into two groups and try two different methods of removing the lux and replacing it with tet. While the other group will be doing the original overlap PCR, our group will be trying restriction enzyme ligation. Both groups will be performing the same basics, but going about the process in different ways. In the end we’re just trying to find which process is more reliable and efficient.   Before we could start doing restriction enzyme ligation, we needed to create more plasmids, as the various experiments over the week have depleted our supply. Using New
Read more