Oct 04, 2024
Sinai SCENT TMC - 5x5 Project Bulk RNAseq
- HIMC at Mount Sinai1
- 1Icahn School of Medicine at Mount Sinai
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: HIMC at Mount Sinai 2024. Sinai SCENT TMC - 5x5 Project Bulk RNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v926w4l3e/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: October 04, 2024
Last Modified: October 04, 2024
Protocol Integer ID: 109194
Abstract
Bulk RNA sequencing protocol
Protocols
Protocols
Dissect the snap-frozen or -80'C stored tissues.
Dissected tissues are placed directly into a labeled 1.5 mL RNAlater (Qiagen) collection tube and stored at 4°C for up to 24 hours to allow the RNAlater to penetrate the tissue fully before being transferred to -20°C or lower until use.
The mRNA is extracted with the RNeasy Plus Mini kit (Qiagen) per the manufacturer’s instructions.
The complementary DNA (cDNA) library is established by using the Ovation RNA-Seq v2 method (NuGen).
Samples are sequenced on an Illumina NovaSeq sequencer with 2X150 bp in the paired ends at 350M read depth per sample.
Gene and isoform expression levels will be calculated using Cufflinks.
Transcripts from mitochondrial and ribosomal RNA genes are masked and not included.
The final abundances of genes in cells are computed in FPKM (Fragments Per Kilobase of exon model per Million mapped fragments).