New England Biolabs, E6420S, NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina®

NEBNext Related Categories RNA Library Prep for Illumina,, Next Generation Sequencing Library Preparation Specification Materials Required but not Supplied 80% Ethanol (freshly prepared) Nuclease-free Water DNA LoBind Tubes (Eppendorf® #022431021) Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent) Thermocycler Vortex Microcentrifuge SPRIselect Reagent Kit (Beckman Coulter®, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881) Agilent® Bioanalyzer® or similar fragment analyzer and associated consumables NEBNext Oligos DNase RNase free PCR strip tubes (USA Scientific 1402-1708) FAQ Q: How much PBS can be carried over from cell collection into the cDNA Synthesis and Amplification protocol? A: For optimal results, PBS carryover should be limited to ≤5µl. Higher carryover volumes are not recommended, and will compromise yield and data quality. Q: What cell types does the NEBNext® Single Cell/Low Input RNA Library Prep Kit support? A: The kit is optimized for use with a variety of eukaryotic cell types. However, the cell lysis is not compatible with fixed cells or cells with cell walls (e.g., plant and fungal cells). If good quality RNA is extracted from these samples, Chapter 2 for low input RNA can be followed for cDNA synthesis, amplification and library generation. The kit is not compatible with mRNA lacking poly(A) tails (e.g., from bacterial cells). Q: Can I resuspend the cells in buffers/medium other than PBS before cell collection? A: We strongly recommend that cells be resuspended in PBS buffer before cell collection. If PBS alone is not a viable option, cells can be resuspended in PBS supplemented with BSA. Up to 0.5 ng of BSA can be included in the cDNA Synthesis and Amplification reaction. If cells are resuspended in cell culture medium, for example, DMEM with 10% FBS, the carry-over volume during cell collection should be minimal (10-15 nl). For sorted cells do not use more than 5 µl carryover volume. Please see this FAQ. Q: Do the cells have to be sorted in to a specific reagent? A: We recommend that the cells be sorted into 1X Cell Lysis Buffer as instructed in the manual. Q: Can frozen cells be used? A: Yes. The cells can be sorted into 1X Cell Lysis Buffer, flash frozen and stored at -80°C. If, instead, the cells are collected in PBS or water before freezing, make the 1X Cell Lysis Buffer according to Section 1.2 (Cell Collection and Lysis) of the manuals supplied with the NEBNext Single Cell/Low Input RNA Library Prep Kit (NEB #E6420) or the NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (NEB #E6421), taking account of the volume the cells are stored in. Q: Can cells be lysed using other cell lysis buffers? A: We do not recommend lysing the cells using cell lysis buffers other than the supplied buffer. The kit is incompatible with cell lysis buffers that contain guanidium salts or denaturing detergents, such as SDS. Q: How long can the synthesized cDNA (prior to cDNA amplification) be stored for prior to amplification? A: The cDNA (prior to cDNA amplification) can be safely stored overnight at 4˚C or -20˚C. If the sample must be stored for a longer time, add 0.5 µl of 10X NEBNext Cell Lysis Buffer to the cDNA (after RT instead of during the PCR step). The samples can then be stored at -20˚C for up to 7 days, but a slight decrease in yield may be observed. Q: What types of RNA are compatible? A: cDNA is selectively synthesized directly from poly(A) tail-containing RNAs, and template switching during reverse transcription is most efficient from template RNA containing a 5´ cap structure. Therefore, the kit works most efficiently with RNA samples with a high RNA Integrity Number (RIN) ≥8. The kit is not suitable for highly fragmented FFPE RNA samples. Q: Does the input total RNA have to be purified? A: The RNA sample should be free of salts (e.g., Mg2+, or guanidinium salts), divalent cation chelating agents (e.g., EDTA, EGTA, citrate), or organics (e.g., phenol and ethanol). If an excess amount of genomic DNA is present in RNA samples, an optional DNase I treatment can be performed. DNase I should be inactivated/removed after treatment. Q: My total RNA sample is in a volume > 8 µl. Can I still use the kit? A: Yes. For total RNA in water or low TE, you can use up to 11 µl by eliminating the addition of water in Section 2.3 (Reverse Transcription and Template Switching). For sorted cells do not use more than 5 µl carryover volume. Please see this FAQ. Q: Are the two PCR master mixes in the kit interchangeable? A: No. The NEBNext Single Cell cDNA PCR Master Mix should be used to amplify cDNA after the RT reaction while the NEBNext Ultra II Q5 Master Mix should be used to amplify the final libraries for Illumina® sequencing. Q: For low cDNA input do I have to do a double bead cleanup after the final PCR? A: For low cDNA input (100-200 pg), it may be necessary to perform a double bead cleanup to remove excess adaptor dimers. To determine library purity, run the libraries on a DNA High Sensitivity Bioanalyzer® chip, or similar instrument. Q: How much cDNA input can I use for the NEBNext Ultra II FS DNA library preparation? A: 100 pg–20 ng purified cDNA. We recommend that the cDNA be in 1X TE (10 mM Tris pH 8.0, 1 mM EDTA); however, 10 mM Tris pH 7.5–8, low EDTA TE or H2O are also acceptable. If the input DNA is less than 26 µl, add TE to a final volume of 26 µl. Q: What is the expected size range of libraries generated with the NEBNext Single Cell/Low Input RNA Library Prep Kit? A: Based on the protocol, the expected Illumina sequencing library size range is 300 bp – 350 bp. Adjust the fragmentation time if different fragment sized libraries are preferred. Q: Why are the adaptor dilution and library amplification PCR cycle number recommendations in this kit different from the NEBNext Ultra II FS DNA Library Prep Kit? A: The recommendations in this kit are optimized for the cDNA input generated from the Single Cell/Low Input cDNA Synthesis & Amplification protocol upstream of the library preparation protocol. Q: Does the NEBNext Single Cell/Low Input RNA Library Prep Kit include adaptors and primers? A: No. Adaptors and primers are supplied separately. Q: How should the adaptor sequences be trimmed? A: The sequences of our oligos used in the workflow are as follows: NEBNext Template Switching Oligo GCT AAT CAT TGC AAG CAG TGG TAT CAA CGC AGA GTA CAT rGrGrG NEBNext Single Cell RT Primer AAG CAG TGG TAT CAA CGC AGA GTA CTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TV NEBNext Single Cell cDNA PCR Primer AAG CAG TGG TAT CAA CGC AGA GT Several trimming tools can be used to trim the reads, which have these sequences on their ends. We recommend Flexbar v3.3.0 or later. Instructions to trim the adaptors using Flexbar can be accessed here: https://github.com/nebiolabs/nebnext-single-cell-rna-seq The typical read length employed for single cell sequencing is 2x75bp but this can be changed depending on insert size distribution. Q: Can I use this NEBNext kit with adaptors and primers from other vendors than NEB? A: Yes, it should be possible to use adaptors and primers from vendors other than NEB with this NEBNext kit. The adaptor must be a T overhang adaptor and the final library yield may be slightly lower than when using the NEBNext adaptors for Illumina®. Instructions for use with non-NEBNext, full length barcoded adaptors and P5 and P7 universal PCR primers can be found in the manuals for the NEBNext indexed/ UMI adaptor manuals. These manuals can be found at neb.com/E7416 (RNA) or neb.com/E7395 (DNA). Choose the kit manual for the NEBNext kit you will be using. The manuals are written specifically for the NEBNext indexed/ UMI adaptors, the same protocol should work with other adaptors of similar design. We recommend performing a proof of principle experiment with the desired library prep kit and desired indexed adaptors and using expendable samples for optimization of experimental conditions. Q: Does the kit include adaptor and primers? A: No. The kit can be used with the NEBNext Multiplex Oligos for Illumina. For a complete list, see NEBNext® Multiplex Oligos Selection Chart. For use with NEBNext library prep kits, consult the library prep kit manual. The multiplex oligo kit specific manuals also contain tips for setting up PCR reactions. For color balancing options, please see NEBNext® Index Oligo Selector for valid barcode combinations.