- NEXTFLEX® Bisulfite-Seq End Repair Buffer Mix
- NEXTFLEX® Bisulfite-Seq End Repair Enzyme Mix
- NEXTFLEX® Bisulfite-Seq Adenylation Mix
- NEXTFLEX® Bisulfite-Seq Ligation Mix
- NEXTFLEX® Bisulfite-Seq DNA Adapter 24 (25 µM)
- NEXTFLEX® Adapter Dilution Buffer
- NEXTFLEX® Primer Mix (12.5 µM)
- NEXTFLEX® Bisulfite-Seq U+ PCR Master Mix
- Nuclease-free water
- Resuspension Buffer
REQUIRED MATERIALS NOT PROVIDED
- 10 ng – 1 µg of fragmented /or/ Msp1 digested genomic DNA in up to 40 µL nuclease-free water.
- NEXTFLEX® Bisulfite-Seq Barcodes – 6 / 12 / 24 (Cat # 511911, 511912, 511913 ) (Optional)
- EZ DNA Methylation-Gold™ Kit (Zymo Research, Cat # D5005)
- Ethanol 100% (room temperature)
- Ethanol 80% (room temperature)
- 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) / or / similar
- 96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) / or / similar
- Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)
- Agencourt AMPure XP 5 mL (Beckman Coulter Genomics, Cat # A63880)
- Magnetic Stand – 96 (Ambion, Cat # AM10027) / or / similar
- Heat block
- 2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes
- Nuclease-free barrier pipette tips
- 1.5 mL nuclease-free microcentrifuge tubes
Papers that Cite the use of the NEXTFLEX® Bisulfite-Seq Kit:
- Agarwal, P., et al. (2015) CGGBP1 mitigates cytosine methylation at repetitive DNA sequences. BMC Genomics, 16:390. doi:10.1186/s12864-015-1593-2.
- Berg, A., et al. (2015) Obesity is Associated With DNA Methylation in Population-Based Adolescents. Circulation. Poster: 131:AP260, Session Title: Obesity.
- Bewick, A., et. al. (2016) On the origin and evolutionary consequences of gene body DNA methylation. PNAS. 113: 9111 – 9116.
- Ramesh, V., et. al. (2016) Loss of Uhrf1 in neural stem cells leads to activation of retroviral elements and delayed neurodegeneration. Genes & Development. 30: 2199 – 2212.
- Satgé, C. et. al. (2016) Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula. Nature Plants 2, 16166. doi:10.1038/nplants.2016.166.
- Ye, R., et al. (2015) A Dicer-Independent Route for Biogenesis of siRNAs that Direct DNA Methylation in Arabidopsis. Molecular Cell. doi:10.1016/j.molcel.2015.11.015.
Do you have protocols available for whole-genome bisulfite sequencing or reduced-representation bisulfite sequencing?
Yes, the NEXTFLEX® Bisulfite-Seq Kit can be used for either WGBS or RRBS.
How much input DNA can be used with the NEXTflex Bisulfite-Seq Kit?
10 ng -1 µg of fragmented genomic DNA (for WGBS) or Msp1 digested genomic DNA (for RRBS) in up to 40 µL nuclease-free water.
How many samples can be multiplexed using the NEXTflex Bisulfite-Seq Kit? Are the indexed adapters available separately?
The methylated NEXTFLEX® Bisulfite-Seq Barcodes are recommended for use with the NEXTFLEX® Bisulfite-Seq Kit. To offer greater flexibility, these indexed adapters are sold separately from the library preparation kits in sets of 6 or 12 unique barcodes. The 8 reaction size of the NEXTFLEX® Bisulfite-Seq Kit (cat # 5119-01) does contain a single adapter for those customers who do not need to multiplex samples. The other barcode kits we offer are not methylated and are not compatible with bisulfite sequencing.
Do you recommend a bisulfite conversion kit?
We recommend the EZ DNA Methylation-Gold™ Kit (Zymo Research, Cat # D5005)
What is the general efficiency of bisulfite conversion using the recommended bisulfite conversion kit?
Conversion efficiency generally exceeds 99% using the EZ DNA Methylation Kit.
The NEXTFLEX® Bisulfite-Seq Kits contain enough material to prepare 8 or 48 genomic DNA samples for Illumina® compatible sequencing. The shelf life of all reagents is 6 months when stored properly. DNA Binding Buffer and DNA Wash Buffer should be stored at room temperature. All of the other components can be safely stored at -20°C.