Jul 12, 2023
JAX SCBL workflow for scRNA-seq of human placenta
- 1The Jackson Laboratory for Genomic Medicine
Protocol Citation: Diane Luo, Jessica Grassmann, Paul Gabriel, William F Flynn, Elise Courtois, Paul Robson 2023. JAX SCBL workflow for scRNA-seq of human placenta. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lynw6pvx9/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: March 16, 2022
Last Modified: July 12, 2023
Protocol Integer ID: 59523
Abstract
Described here is the workflow used by the JAX Single Cell Biology lab to generate single cell transcriptomic libraries from human placenta samples collected at the UCSD HuBMAP Female Reproductive Tissue Mapping Center.
Materials
See accompanying protocols.
Tissue collection
Tissue collection
Tissue is collected and processed as soon as possible after Cesarean section or vaginal delivery according to the following protocol:
Tissue is then stored in MACS storage buffer for overnight shipping.
Sample preparation
Sample preparation
Upon receiving the sample, single cells are isolated through a digestion with cold active protease as described here:
The viability and cell concentration for each sample is assessed via a Countess FL II or LUNA FX7 automated cell counter.
The single cell suspension is then loaded onto one lane of the appropriate 10x Genomics gene expression chip as follows:
| A | B | C | D | |
| Chip | Approx Cells Loaded | Approx. Cells Targeted | Compatiable Instrument | |
| Chip G | 12,000 | 6,000 | Chromium Controller, Xi, X | |
| Chip M | 40,000 | 20,000 | Chromium X |
Library Generation
Library Generation
The resulting loading, running, and library construction is performed according to the appropriate 10x Genomics protocol:
Sequencing
Sequencing
We have typically targeted 100,000 read pairs/cell for human placenta sample, resulting in target library sizes of roughly 600M-2,000M read pairs. These are multiplexed on a NovaSeq 6000 S4 lane using v1.5 reagents and the reverse complement workflow in a 28/8/0/90 or 28/8/8/90 configuration.