Jul 15, 2023
Workflow for bulk RNAseq of human fallopian tube and uterine endomyometrium
- Scott Lindsay-Hewett1,
- Valentina Stanley1,
- Mana Parast1,
- Louise Laurent1
- 1University of California, San Diego
Protocol Citation: Scott Lindsay-Hewett, Valentina Stanley, Mana Parast, Louise Laurent 2023. Workflow for bulk RNAseq of human fallopian tube and uterine endomyometrium . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l224p3l1y/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 15, 2023
Last Modified: July 15, 2023
Protocol Integer ID: 85043
Abstract
Described here is the workflow used by the Female Reproductive Tissue Mapping Center at UCSD to generate bulk RNAseq data from human fallopian tube and uterine endomyometrium.
Tissue preparation
Tissue preparation
As soon as possible after sterilization (salpingectomy or tubal ligation), prepare fallopian tube tissue according to the following protocol:
At the time of C-section, prepare uterine endomyometrium tissue according to the following protocol:
For this protocol, use tissue that has been collected in RNAlater.
Total RNA isolation
Total RNA isolation
Isolate total RNA using a bead beater to disrupt the tissue, followed by organic extraction and ethanol precipitation. Use the following protocol, which was originally written for placenta:
After passing quality control, proceed to library construction.
Note
In our hands, RIN scores for fallopian tube and uterine endomyometrium were quite low. Bioanalyzer DV200 scores (the percentage of fragments >200 nucleotides) were instead used to assess RNA quality, and libraries were constructed according to a ribodepletion method which is more appropriate for low-quality RNA.
Library construction
Library construction
Construct libraries using the KAPA RNA HyperPrep Kit with RiboErase (HMR), according to the following protocol, which was originally written for placenta:
Note
The following adaptations were made to the above protocol:
Fragmentation: tailored to DV200 score (DV200>=70, 00:05:00 85 °C ; DV200>=60, 00:04:00 85 °C ; DV200>=50, 00:03:00 85 °C ; DV200>=40, 00:02:00 85 °C ; DV200>=30, 00:01:00 85 °C ; DV200<30, 00:01:00 65 °C ).
Adapter concentration: 1.5 micromolar (µM)
Library amplification: Pre-amplification library concentrations were assessed using the Qubit High Sensitivity DNA assay. The following formula was used, where x = pre-amplification library concentration (in ng/ul), to estimate the number of amplification cycles (rounded up to the next cycle) required for each library:
Number of amplification cycles = 5 + log2(3/x)
After passing quality control, proceed to sequencing.
Sequencing
Sequencing
For HuBMAP bulk RNAseq samples, the multiplexed pool was sequenced on a NovaSeq 6000 S4 lane using a 100bp paired-end run configuration. Reads were aligned using STAR, and transcript abundances were quantified using RSEM.