Mar 14, 2022
Workflow for bulk RNAseq of human placenta
- 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 2022. Workflow for bulk RNAseq of human placenta. protocols.io https://dx.doi.org/10.17504/protocols.io.b59sq96e
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: March 14, 2022
Last Modified: March 14, 2022
Protocol Integer ID: 59410
Abstract
Described here is the workflow used by the Female Reproductive Tissue Mapping Center at UCSD to generate RNAseq data from human placenta.
Materials
See accompanying protocols.
Tissue preparation
Tissue preparation
As soon as possible after Cesarean section or vaginal delivery, prepare tissue according to the following protocol:
For this protocol, use tissue that has been stored 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:
After passing quality control, proceed to library construction.
Library construction
Library construction
Construct libraries using the KAPA RNA HyperPrep Kit with RiboErase (HMR), according to the following protocol:
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.