Jun 17, 2022
Workflow for human fallopian tube and uterine endomyometrium bulk ATACseq
- 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 human fallopian tube and uterine endomyometrium bulk ATACseq. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl898brv2w/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: June 17, 2022
Last Modified: June 17, 2022
Protocol Integer ID: 64752
Abstract
Described here is the workflow used by the Female Reproductive Tissue Mapping Center at UCSD to generate bulk ATACseq data from human fallopian tube and uterine endomyometrium.
Materials
See accompanying protocols.
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 snap-frozen.
Nuclei isolation
Nuclei isolation
Isolate nuclei from 4 samples at a time according to the following protocol:
Proceed to tagmentation immediately.
Note
Protocol above was written for human placental tissue but can be used for fallopian tube and uterine endomyometrium. Isolated nuclei are paler than those from placenta and should be counted manually with a hemocytometer instead of using an automated cell counter which can struggle to detect them. Nuclei also have a different morphology to placenta, and are much sparser in uterine endomyometrium.
Representative image of nuclei isolated from fallopian tube.
Representative image of nuclei isolated from uterine endomyometrium.
Tagmentation and library generation
Tagmentation and library generation
Perform tagmentation and library generation according to the following protocol:
After passing quality control, proceed to sequencing.
Note
Protocol above was written for human placental tissue but can be used for fallopian tube and uterine endomyometrium. Bioanalyzer traces typically show a less obvious nucleosomal laddering pattern when compared to those from placenta.
Typical Bioanalyzer trace for a fallopian tube bulk ATACseq library.
Typical Bioanalyzer trace for a uterine endomyometrium bulk ATACseq library.
Sequencing
Sequencing
For HuBMAP bulk ATACseq samples, the multiplexed pool was sequenced on a NovaSeq 6000 S4 lane using a 100bp paired-end run configuration. Alignment and peak-calling were performed using the ATAC-seq pipeline within the bcbio Python package.