Nov 11, 2025
JAX-Sen: Watchmaker RNA Library Prep Kits with Polaris Depletion
- Yongjun Huang1,
- Chrystal Snow1,
- Jenn Idol1,
- Qingchang Meng1,
- Chew Yee Ngan1,
- Mark Adams1
- 1The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: Yongjun Huang, Chrystal Snow, Jenn Idol, Qingchang Meng, Chew Yee Ngan, Mark Adams 2025. JAX-Sen: Watchmaker RNA Library Prep Kits with Polaris Depletion. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnrybzl5d/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: October 14, 2025
Last Modified: November 11, 2025
Protocol Integer ID: 229827
Keywords: watchmaker rna library prep kits with polaris depletion, rna library prep kit with polaris depletion protocol, watchmaker rna library prep kit, rna library prep kit, polaris depletion protocol, polaris depletion, library preparation
Funders Acknowledgements:
National Institute on Aging (NIA) JAX-Sen Senescence Tissue Mapping Center
Grant ID: U54 AG079753
Abstract
This protocol describes the library preparation using the RNA Library Prep Kit with Polaris Depletion protocol followed by sequencing on the Illumina NovaSeq X-Plus platform using patterned flow cells and two-color chemistry.
Troubleshooting
Materials and Reagents:
Instruments:
- Qubit Flex Fluorometer, Thermo Fisher Scientific
- TapeStation 4200, Agilent Technologies
- Thermal Cycler, Bio-Rad
- NovaSeq X-Plus, Illumina
Procedure:
The concentration and integrity of RNA and sequencing libraries were assessed using the Qubit Flex Fluorometer (Thermo Fisher Scientific) and the Agilent TapeStation 4200 (Agilent Technologies).
Library preparation:
RNA-Seq libraries were prepared using the RNA Library Prep Kit with Polaris Depletion (Watchmaker, Cat# 7BK0002-096) following a modified version of the manufacturer’s protocol (Watchmaker protocol). Modifications included the addition of ERCC spike-in to each sample, a 0.8× bead clean-up after ligation, and a 0.7× bead clean-up after PCR amplification.
Indexes used were Illumina xGen‱ UDI-UMI Adapters, 1-96 (10005903) and special orders from IDT.
Sequencing:
Sequencing was performed on the Illumina NovaSeq X-Plus platform using patterned flow cells and two-color chemistry (green and blue channels). Libraries were sequenced on either a 10B or 25B flow cell, each containing eight lanes. Runs were carried out with 300-cycle kits, which include an additional 38 cycles for index reads. The expected yield per lane is approximately 1,250 million reads on a 10B flow cell and 3,250 million reads on a 25B flow cell.