Workflow for bulk RNAseq of human placenta
Scott Lindsay-Hewett, Valentina Stanley, Louise Laurent, Mana Parast
Abstract
Described here is the workflow used by the Female Reproductive Tissue Mapping Center at UCSD to generate RNAseq data from human placenta.
Steps
Tissue preparation
As soon as possible after Cesarean section or vaginal delivery, prepare tissue according to the following protocol:
Human Placenta Tissue Collection and Preservation Methods - UCSD Female Reproductive TMC
For this protocol, use tissue that has been stored in RNAlater.
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:
Total RNA extraction from frozen placenta tissue
After passing quality control, proceed to library construction.
Library construction
Construct libraries using the KAPA RNA HyperPrep Kit with RiboErase (HMR), according to the following protocol:
Library construction for human placenta bulk RNAseq
After passing quality control, proceed to 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.
