Jul 16, 2023

Public workspaceBulk RNA sequencing

DOI
dx.doi.org/10.17504/protocols.io.rm7vzx362gx1/v1
  • 1Columbia University
Connor Monahan
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzx362gx1/v1
Protocol Citation: Connor Monahan 2023. Bulk RNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx362gx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 12, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84902
Keywords: ileum, bulk RNA sequencing, gut, small intestine, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: 0375
Abstract
This protocol details bulk RNA sequencing from the mouse small intestine.
Attachments
Icon for 786-2002.pdf
786-2002.pdf
47KB
Materials
Materials

  • PBS
  • Illumina NovaSeq6000

ReagentTRIzol ReagentThermo Fisher ScientificCatalog #15596026


Procedure
Procedure
Collect the ileum from a PBS-perfused mouse.
Thoroughly flush the ileum sample with cold 1x PBS.
Incubate samples in TRIzol reagent (Thermo Fisher Scientific, Cat #15596026; Waltham, MA) and store at Temperature-80 °C until ready for bulk RNA sequencing.

Incubation
Extract RNA and construct library using Illumina TruSeq chemistry.
Sequence the libraries using an Illumina NovaSeq6000.
Samples are multiplexed in each lane, which yielded targeted number of paired-end, 100bp reads for each sample. RTA (Illumina) for base calling and bcl2fastq2 (version 2.19) are used for converting BCL to fastq format, coupled with adaptor trimming.
Perform a pseudoalignment to a kallisto index created from transcriptomes (GRCm38) using kallisto (0.44.0).
Test differentially expressed genes under various conditions using DESeq2R packages designed to test differential expression between two experimental groups from RNA-seq counts data.
Genes are considered differentially expressed if they had an adjusted p-value <0.05 and a log2fold change below or above 0.5. Normalize the differential expression for each gene.