Total RNA from cells (U-clones) was extracted using TRIzol (Invitrogen 15596018). mRNAs were enriched using Oligo d(T)25 Magnetic Beads (NEB S1419S) following manufacturer’s protocol. mRNAs were then fragmented at 94°C in 10 mM MgCl2 buffer. Fragmented RNAs were end-repaired by T4 PNK (NEB M0201S) and poly(A)-tailed by E. coli Poly(A) Polymerase (NEB M0276S). The poly(A) tailed RNA fragments were reverse transcribed to cDNA using custom-designed oligo(dT) and locked nucleic acid (LNA) based on SMART-seq2 method (Picelli et al., 2014 (link)). The cDNAs were then PCR amplified using KAPA HiFi HotStart master mix (KK2601). 200-400 bp fragments of the library were cut from gels for Illumina Hiseq sequencing.
Kapa hifi hotstart master mix
Manufactured by Roche
Sourced in China, United States
KAPA HiFi HotStart master mix is a high-fidelity PCR reagent designed for sensitive and accurate DNA amplification. It provides robust and reliable performance for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq sequencing, by Illumina (1 mentions)
E coli poly a polymerase, by New England Biolabs (1 mentions)
Oligo d t 25 magnetic beads, by New England Biolabs (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
T4 pnk, by New England Biolabs (1 mentions)
Evagreen, by Biotium (1 mentions)
Nebnext multiplex oligos for kit, by Illumina (1 mentions)
5 mc monoclonal antibody, by Diagenode (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Novaseq platform, by Illumina (1 mentions)
Magmedip kit, by Diagenode (1 mentions)
4 protocols using kapa hifi hotstart master mix
1
Transcriptome Profiling of U-clones
2
cDNA Amplification and Purification
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Wash beads and resuspend.
Wash once with 400 μL of 10 mM Tris and 0.1% Tween-20 solution and once more with 400 μL of RNase-free water.
Resuspend in the PCR mix solution containing 110 μL of 2× Kapa HiFi HotStart Master Mix (Kapa Biosystems), 8.8 μL each of 10 μM stocks of primers 1 and 2, and 92.4 μL of RNase-free water. Transfer 200 μL to four PCR tubes with 50 μL in each.
Perform PCR to detach cDNA from beads.
| PCR cycling conditions | |||
|---|---|---|---|
| Steps | Temperature | Time | Cycles |
| Initial Denaturation | 95°C | 3 min | 1 |
| Denaturation | 98°C | 20 s | 5 cycles |
| Annealing | 65°C | 45 s | |
| Extension | 72°C | 3 min | |
Remove the DynabeadsTM from the PCR solution using a magnetic tube holder.
Add Evagreen (Biotium) at a 1× concentration.
Perform PCR again with the following thermocycling conditions. Cycling can also be halted once the qPCR signal begins to plateau.
| PCR cycling conditions | |||
|---|---|---|---|
| Steps | Temperature | Time | Cycles |
| Initial Denaturation | 95°C | 3 min | 1 |
| Denaturation | 98°C | 20 s | 15 cycles |
| Annealing | 65°C | 20 s | |
| Extension | 72°C | 3 min | |
| Final Extension | 72°C | 5 min | 1 |
| Hold | 4°C | Forever | |
3
cfDNA Methylation Profiling by cfMeDIP-seq
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cfDNA extracted from plasma was then used for cfMeDIP-seq library preparation with the method described previously with the following modifications [3 (link), 44 (link)].
(1) ~ 10 to 20 ng cfDNA was ligated with a pool of eight unique adapters with 8-bp molecular barcodes instead of the single adapter (NEBNext Multiplex Oligos for Illumina kit, New England BioLabs) (Additional file1 : Table S6), each initial cfDNA fragment was labeled with a unique barcode. The ligation was conducted by using KAPA Hyper Prep kit (KAPA biosystems, KK8504) according to manufacturer’s instructions; (2) The 5-mC monoclonal antibody (Diagenode, C02010021) immunoprecipitated cfDNA and input cfDNA were amplified using Kapa HiFi Hotstart Mastermix (KAPA biosystems, KK8504) and oligos listed in Additional file 1 : Table S6; (3) The multiplexed libraries were subjected for BioAnlyzer analysis before sequencing on Illumina Novaseq platform at HaploX (Shenzhen, China) with 2 × 150-bp paired-end (PE) reads; (4) Input and IP libraries were sequenced at 0.5 × and 5 × respectively.
The specificity of the immunoprecipitation reaction and fold-enrichment ratio in IP libraries were evaluated using the MagMeDIP kit (Diagenode, C02010021) according to the manufacturer’s instructions.
(1) ~ 10 to 20 ng cfDNA was ligated with a pool of eight unique adapters with 8-bp molecular barcodes instead of the single adapter (NEBNext Multiplex Oligos for Illumina kit, New England BioLabs) (Additional file
The specificity of the immunoprecipitation reaction and fold-enrichment ratio in IP libraries were evaluated using the MagMeDIP kit (Diagenode, C02010021) according to the manufacturer’s instructions.
4
Yeast Display Library Screening
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Sorted yeast were expanded at 30°C for 24–48 h after each round of FACS screening. A portion of this culture was used for high-efficiency yeast plasmid DNA extraction (47 (link)). A high-fidelity polymerase (Kapa Hifi HotStart Mastermix, Kapa Biosystems, Massachusetts, USA) and primers targeting the yeast display vector backbone (Table S4
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