Mar 25, 2020
- Graham J Etherington1,
- Darren Heavens1,
- David Baker1,
- Ashleigh Lister1,
- Rose McNelly1,
- Gonzalo Garcia1,
- Bernardo Clavijo1,
- Iain Macaulay1,
- Wilfried Haerty1,
- Federica Di Palma1
- 1The Earlham Institute, Norwich Research Park, Norwich, NR4 7UZ, United Kingdom
- GigaScience Press
External link: https://doi.org/10.46471/gigabyte.4
Protocol Citation: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma 2020. 10x Genomics Library Construction. protocols.io https://dx.doi.org/10.17504/protocols.io.bd3vi8n6
Manuscript citation:
Weixue Mu, Jinpu Wei, Ting Yang, Yannan Fan, Le Cheng, Jinlong Yang, Ranchang Mu, Jie Liu, Jianming Zhao, Weibang Sun, Xun Xu, Xin Liu, Radoje Drmanac, Huan Liu, The draft genome assembly of the critically endangered Nyssa yunnanensis, a plant species with extremely small populations endemic to Yunnan Province, China, Gigabyte, 2020 https://doi.org/10.46471/gigabyte.4
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 23, 2020
Last Modified: August 14, 2020
Protocol Integer ID: 34645
Keywords: polecat, vertebrate, non-model organism, Illumina, chromium, Bionano, assembly, sequencing,
Abstract
10x Genomics Library Construction
Input DNA (in this case Polecat) was diluted down from 4.18 ng/ul to 1 ng/ul using Qiagen EB and checked using a QuBit 2.0 Fluorometer (Thermo-Fisher). The sample was then diluted in half as it was denatured. Finally, 2.5 µL of the diluted and denatured DNA was loaded onto the 10X chip which equated to 1.25 ng .
Gel beads, oil and master mix were loaded into the appropriate wells on the 10X chip. The DNA was loaded along with the master mix. The chip was then loaded into the 10X Genomics Chromium instrument.
The formed emulsion was removed and placed into the thermocycler for 03:00:00 at 30 °C . GEMs were cleaned up using the recovery reagent (10X Genomics), Silane beads (Thermo Fisher) and Tween-20 (Sigma-Aldrich) as per manufacturer’s instructions. DNA is cleaned up using a 0.7X ratio of SPRISelect beads (Beckman Coulter).
The library underwent simultaneous End Repair and A tailing on the thermocycler at 20 °C for 00:30:00 and then 65 °C for 00:30:00 . Adapters were then ligated at 20 °C for 00:15:00 . A 0.8X ratio SPRISelect clean-up was performed. Sample Indexes were added during the final 8 cycle PCR amplification.
| Temperature | Time | |
| 98°C (step 1) | 45 secs | |
| 98°C (step 2) | 20 secs | |
| 54°C (step 3) | 30 secs | |
| 72°C (step 4) | 20 secs | |
| Steps 2-4 for 7 or 8 cycles in total | ||
| 72°C (step 5) | 60 secs | |
| 4°C (step 6) | Hold |
A dual-SPRI size selection was performed at 0.5X then 0.7X ratios.
Library sizes were checked using the High Sense DNA bioanalyzer (Agilent). Molarity was checked using qPCR (KAPA Library Quant kit (Illumina), using ABI Prism qPCR Mix (Kapa Biosystems).
The library was clustered on the flow cell using the qPCR molarity. It was clustered at 8pM along with 1% PhiX spike-in (Illumina). Two lanes of a Rapid Run v2 flow cell were run on an Illumina HiSeq2500. 150bp PE, 8bp i7 index read, 0bp i5 index read. The libraries clustered at around 1100K/mm2.
Manufacturer’s instructions: https://assets.contentful.com/an68im79xiti/4z5JA3C67KOyCE2ucacCM6/d05ce5fa3dc4282f3da5ae7296f2645b/CG00022_GenomeReagentKitUserGuide_RevC.pdf