Jan 28, 2022
Library construction for human placenta bulk RNAseq
- 1University of California, San Diego
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: Jeff.spraggins@vanderbilt.edu
Protocol Citation: Scott Lindsay-Hewett 2022. Library construction for human placenta bulk RNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.b4fyqtpw
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: January 28, 2022
Last Modified: January 28, 2022
Protocol Integer ID: 57560
Abstract
This protocol describes the generation of stranded RNA-seq libraries from placenta total RNA. Since RNA quality can be an issue with total RNA isolated from placental tissue, it is advisable to enrich mRNA using a ribodepletion method rather than polyA selection.
Prior to ribodepletion, it is especially important to ensure that the input RNA is free of contaminating DNA. This protocol, therefore, begins with DNase treatment of samples, using Ambion's DNA-free DNase Treatment and Removal Reagents. Next, ribodepletion and library construction are performed using the KAPA RNA HyperPrep Kit with RiboErase (HMR). Libraries are indexed with KAPA Unique Dual-Indexed Adapters to enable multiplexed sequencing on an Illumina instrument.
Materials
Equipment
Veriti 96-Well Thermal Cycler
NAME
Applied Biosystems
BRAND
4375786
SKU
LINK
Equipment
Qubit
NAME
Flurometer
TYPE
Invitrogen
BRAND
Q33228
SKU
LINK
Equipment
Centrifuge
NAME
Benchtop Centrifuge
TYPE
Eppendorf
BRAND
5405000441
SKU
LINK
Any benchtop centrifuge will suffice
SPECIFICATIONS
Equipment
Set of micropipettes with rack: 100-1000 µl, 20-200 µl, 2-20 µl, and 0.5-10 µl
NAME
Pipettor set
TYPE
Pipetman
BRAND
QP-1001-07
SKU
LINK
Can use equivalent Pipettors
SPECIFICATIONS
Equipment
Vortex mixer
NAME
Any
BRAND
xx
SKU
Equipment
Magnetic Stand
NAME
Magnetic Stand
TYPE
Thermo Scientific
BRAND
MR02
SKU
LINK
Any magnetic rack that fits your tubes will suffice.
SPECIFICATIONS
Equipment
Bioanalyzer
NAME
Bioanalyzer
TYPE
Agilent
BRAND
G2991AA
SKU
LINK
Any bioanalyzer will suffice.
SPECIFICATIONS
Nuclease-free WaterContributed by users
1M Tris pH 8.0Sigma AldrichCatalog #T2694
200 Proof Ethanol pureSigma AldrichCatalog #E7023
Qubit RNA BR Assay KitThermo Fisher ScientificCatalog #Q10211
Qubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854
Agilent High Sensitivity DNA KitAgilent TechnologiesCatalog #5067-4626
DNA-free DNA Removal kitInvitrogenCatalog #AM1906
KAPA RNA HyperPrep Kit with RiboErase (HMR)Kapa BiosystemsCatalog #KK8560 (24 libraries) or KK8561
KAPA Unique Dual-Indexed Adapter KitKapa BiosystemsCatalog #KK8727
DNA removal
DNA removal
To remove any possible contaminating DNA prior to ribodepletion, follow Ambion's protocol (Publication # 1906M, Revision E) for their DNA-free DNase Treatment and Removal Reagents.
DNA-free™ Kit DNase Treatment and Removal Reagents User Guide.pdf Note
Follow instructions for routine DNase treatment.
Tip: when transferring supernatant during the final step, be sure not to draw up any of the DNase Inactivation Reagent. Take off a volume that you are comfortable with. There is practically zero loss of RNA using this method, only what you leave behind. If you need to maximize your RNA yield you can always increase the starting volume by adding nuclease-free water, but be sure that the RNA will still be at least 100 ng/ul for library construction in the next step.
Tip: The DNase Inactivation Reagent settles very quickly, so it is important to follow the instruction to resuspend it immediately before use, and several times during incubation with the reaction mix.
Quantitate DNase-treated total RNA using Qubit RNA Broad Range Assay.
Ribodepletion and library construction
Ribodepletion and library construction
10m 30s
10m 30s
Follow KAPA's protocol (KR1351 - v4.21) for their KAPA RNA HyperPrep Kit with RiboErase (HMR). In our hands, this protocol yields great results with partially degraded total RNA samples from placenta, some with RIN scores as low as 2.
KAPA RNA HyperPrep Kit with RiboErase (HMR) KR1351 - v4.21.pdf For HuBMAP samples, the following parameters were used:
Input total RNA: 1000 ng
Fragmentation: tailored to RIN score (RIN score 6.0 and above, 00:04:00 85 °C ; RIN score 5.0-5.9, 00:03:30 85 °C ; RIN score 4.9 and below, 00:03:00 85 °C )
Library amplification: 8 cycles
For indexing, and to enable efficient multiplexed sequencing, use the KAPA Unique Dual-Indexed Adapter Kit , and dilute adapters to 7 micromolar (µM) . Using UDIs reduces "index hopping" which can be an issue when using combinatorial dual indexes, especially on patterned flow cells like the NovaSeq.
KAPA Unique Dual-Indexed Adapter Kit KR1736 - v3.20.pdf Note
Be extremely careful when pipetting out of the adapter plate - you do not want to cross-contaminate wells. Make sure to centrifuge the adapter plate before carefully peeling off the foil cover. Promptly replace with a fresh foil cover when done. Use provided KAPA Adapter Dilution Buffer to dilute adapters from 15 micromolar (µM) to 7 micromolar (µM) .
Note
If using a thermocycler to incubate the adapter ligation reaction at 20 °C , do not pre-heat the lid as this may cause the tube/plate temperature to be too warm, allowing DNA ends to breathe and reducing adapter ligation efficiency.
10m 30s
Quality control
Quality control
Quantitate libraries using the Qubit DNA High Sensitivity Assay, and check library distribution by running the DNA High Sensitivity Assay on an Agilent Bioanalyzer. A typical trace is shown below.
Expected result
Note the appearance of several primer dimer peaks at ~40-60bp which are present due to a combination of the high concentration of primers in the 10x Library Amplification Primer Mix, and the low number of amplification cycles performed (low number of PCR cycles is preferable to reduce amplification bias). These primer dimers are not an issue since they cannot cluster on the flow cell.
Pay attention to the presence of any adapter dimers, which will appear at ~120bp. These will cluster on the flow cell and reduce the number of reads obtained in a sequencing run. If adapter dimers are present, perform another 1x bead-based cleanup using KAPA Pure Beads.
If multiplexing samples, first perform a balancing run to ensure equal representation of all samples in the pool. For the balancing run, prepare an "equal volume" pool by combining 2 µL each library together. Run the pool on a MiSeq instrument using a MiSeq Reagent Kit Nano. Based on the proportion of reads assigned to each index during the Nano run, prepare a balanced pool that will yield an equal read depth for all samples in the pool.
Prior to submitting for sequencing, quantitate the pool using the Qubit High Sensitivity DNA assay. Determine the average fragment size for each library from the Bioanalyzer traces.
Note
Tip: set a region from 200bp to 1000bp in the region table tab (fragments over ~1kb don't cluster efficiently on the flow cell) and the Agilent software will calculate the average fragment size.
Determine the average fragment size in the balanced pool, and use the following formula to determine the nM concentration:
Submit the pool to your sequencing facility, noting the nM concentration.
For HuBMAP bulk RNA-seq samples, the multiplexed pool was sequenced on a NovaSeq 6000 S4 lane using a 100bp paired-end run configuration.