NEBNext^® Multiplex Small RNA Library Prep Set for Illumina^® (Set 1)

The following reagents are supplied with this product:

NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration
E7300S     -20       NEBNext 3´ Ligation Reaction Buffer E7301AVIAL -20 1 x 0.24 ml Not Applicable   Murine RNase Inhibitor E7308AVIAL -20 1 x 0.024 ml Not Applicable   NEBNext 5´ Ligation Reaction Buffer E7304AVIAL -20 1 x 0.024 ml Not Applicable   NEBNext 5´ Ligation Enzyme Mix E7305AVIAL -20 1 x 0.06 ml Not Applicable   LongAmp™ Taq 2X Master Mix E7309AVIAL -20 1 x 1.2 ml Not Applicable   Quick-Load pBR322 DNA-MspI Digest E7323AVIAL -20 1 x 0.24 ml Not Applicable   DNA Gel Elution Buffer E7324AVIAL -20 1 x 12 ml Not Applicable   Linear Acrylamide E7325AVIAL -20 1 x 0.048 ml Not Applicable   Nuclease-free Water E7327AVIAL -20 1 x 5 ml Not Applicable   TE Buffer E7326AVIAL -20 1 x 0.48 ml Not Applicable   NEBNext Index 1 Primer for Illumina E7311AVIAL -20 1 x 0.01 ml 10 µM   NEBNext Index 2 Primer for Illumina E7312AVIAL -20 1 x 0.01 ml 10 µM   NEBNext Index 3 Primer for Illumina E7313AVIAL -20 1 x 0.01 ml 10 µM   NEBNext Index 4 Primer for Illumina E7314AVIAL -20 1 x 0.01 ml 10 µM   NEBNext Index 5 Primer for Illumina E7315AVIAL -20 1 x 0.01 ml 10 µM   NEBNext Index 6 Primer for Illumina E7316AVIAL -20 1 x 0.01 ml 10 µM   NEBNext Index 7 Primer for Illumina E7317AVIAL -20 1 x 0.01 ml 10 µM   NEBNext Index 8 Primer for Illumina E7318AVIAL -20 1 x 0.01 ml 10 µM   NEBNext Index 9 Primer for Illumina E7319AVIAL -20 1 x 0.01 ml 10 µM   NEBNext Index 10 Primer for Illumina E7320AVIAL -20 1 x 0.01 ml 10 µM   NEBNext Index 11 Primer for Illumina E7321AVIAL -20 1 x 0.01 ml 10 µM   NEBNext Index 12 Primer for Illumina E7322AVIAL -20 1 x 0.01 ml 10 µM   Gel Loading Dye, Blue E6138AVIAL -20 1 x 0.2 ml 6 X   NEBNext 3´ SR Adaptor for Illumina E7332AVIAL -20 1 x 0.024 ml Not Applicable   NEBNext SR RT Primer for Illumina E7333AVIAL -20 1 x 0.024 ml Not Applicable   NEBNext SR Primer for Illumina E7310AVIAL -20 1 x 0.06 ml Not Applicable   NEBNext 5´ SR Adaptor for Illumina E7328AVIAL -20 1 x 1,350 pmol Not Applicable   NEBNext First Strand Synthesis Reaction Buffer E7334AVIAL -20 1 x 0.192 ml Not Applicable   ProtoScript II Reverse Transcriptase E7355AVIAL -20 1 x 0.024 ml Not Applicable   NEBNext 3´ Ligation Enzyme Mix E7288AVIAL -20 1 x 0.072 ml Not Applicable
E7300L     -20       NEBNext 3´ Ligation Reaction Buffer E7301AAVIAL -20 1 x 0.96 ml Not Applicable   Murine RNase Inhibitor E7308AAVIAL -20 1 x 0.096 ml Not Applicable   NEBNext 5´ Ligation Reaction Buffer E7304AAVIAL -20 1 x 0.096 ml Not Applicable   NEBNext 5´ Ligation Enzyme Mix E7305AAVIAL -20 1 x 0.24 ml Not Applicable   LongAmp™ Taq 2X Master Mix E7309AAVIAL -20 4 x 1.2 ml Not Applicable   Quick-Load pBR322 DNA-MspI Digest E7323AAVIAL -20 1 x 0.96 ml Not Applicable   DNA Gel Elution Buffer E7324AAVIAL -20 1 x 48 ml Not Applicable   Linear Acrylamide E7325AAVIAL -20 1 x 0.192 ml Not Applicable   Nuclease-free Water E7327AAVIAL -20 1 x 20 ml Not Applicable   TE Buffer E7326AAVIAL -20 1 x 1.92 ml Not Applicable   NEBNext Index 1 Primer for Illumina E7311AAVIAL -20 1 x 0.04 ml 10 µM   NEBNext Index 2 Primer for Illumina E7312AAVIAL -20 1 x 0.04 ml 10 µM   NEBNext Index 3 Primer for Illumina E7313AAVIAL -20 1 x 0.04 ml 10 µM   NEBNext Index 4 Primer for Illumina E7314AAVIAL -20 1 x 0.04 ml 10 µM   NEBNext Index 5 Primer for Illumina E7315AAVIAL -20 1 x 0.04 ml 10 µM   NEBNext Index 6 Primer for Illumina E7316AAVIAL -20 1 x 0.04 ml 10 µM   NEBNext Index 7 Primer for Illumina E7317AAVIAL -20 1 x 0.04 ml 10 µM   NEBNext Index 8 Primer for Illumina E7318AAVIAL -20 1 x 0.04 ml 10 µM   NEBNext Index 9 Primer for Illumina E7319AAVIAL -20 1 x 0.04 ml 10 µM   NEBNext Index 10 Primer for Illumina E7320AAVIAL -20 1 x 0.04 ml 10 µM   NEBNext Index 11 Primer for Illumina E7321AAVIAL -20 1 x 0.04 ml 10 µM   NEBNext Index 12 Primer for Illumina E7322AAVIAL -20 1 x 0.04 ml 10 µM   Gel Loading Dye, Blue E6138AAVIAL -20 1 x 1 ml 6 X   NEBNext 3´ SR Adaptor for Illumina E7332AAVIAL -20 1 x 0.096 ml Not Applicable   NEBNext SR RT Primer for Illumina E7333AAVIAL -20 1 x 0.096 ml Not Applicable   NEBNext SR Primer for Illumina E7310AAVIAL -20 1 x 0.24 ml Not Applicable   NEBNext 5´ SR Adaptor for Illumina E7328AVIAL -20 1 x 1,350 pmol Not Applicable   NEBNext First Strand Synthesis Reaction Buffer E7334AAVIAL -20 1 x 0.768 ml Not Applicable   ProtoScript II Reverse Transcriptase E7355AAVIAL -20 1 x 0.096 ml Not Applicable   NEBNext 3´ Ligation Enzyme Mix E7288AAVIAL -20 1 x 0.288 ml Not Applicable

1. Kit Optimization
   
   The components of this kit were collectively optimized with FirstChoice®Human Brain Reference RNA (Life Technologies, Inc. #AM7962).
   
   High quality total RNA should be used as starting material. An RNA IntegrityNumber (RIN) > 7 is recommended.
   
   Assess the quality and quantity of your sample using the Agilent 2100Bioanalyzer using an Agilent RNA 6000 Nano Chip.

1. Protocol for use with NEBNext Small RNA Library Prep Set for Illumina (E7300, E7580, E7560, E7330)
2. 6A. Quality Control Check and Size Selection using 6% Polyacrylamide Gel  NEBNext Small RNA Library Prep Set for Illumina (E7300, E7580, E7560, E7330)
3. 6B. Quality Control Check and Size Selection using Pippin Prep NEBNext Small RNA Library Prep Set for Illumina (E7300, E7580, E7560, E7330)
4. 6C. Quality Control Check and Size Selection using AMPure XP Beads NEBNext Small RNA Library Prep Set for Illumina (E7300, E7580, E7560, E7330)

• Automated size selection of NEBNext^® Small RNA libraries with the Sage Pippin Prep™

1.  Is it possible to prevent abundant, unwanted RNAs to be included in the Small RNA library?
2. Can I use Total RNA to make small RNA libraries or do I have to isolate or enrich the sample for small RNA?
3. Why does the RT primer hybridization occur before 5’adaptor ligation?
4. Do I have to hybridize the RT primer again after 5’ ligation?
5. How are the barcodes introduced in the Multiplex libraries?
6. During size selection on 6% PAGE gel, which bands should I cut out of the gel?
7. Are libraries prepared with the NEBNext^® Small RNA Library prep kit for Illumina compatible with paired-end flow cells for cluster generation?
8. Can I use the small RNA sample preparation kit for Directional-RNA sequencing?
9. What is the sequence of the final PCR product?
10. Can I use enriched small RNA instead of total RNA for the NEBNext Small RNA Library Prep for Illumina?
11. What should I do if my Small RNA may not have 5´ Phosphate and 3´ OH groups?
12. How should my NEBNext Small RNA library be trimmed?
13. Are the Monarch Spin RNA Cleanup Kits (NEB #T2030, #T2040, #T2050) compatible with NEBNext reagents for RNA library prep?
14. What should I do if the sample sheet from the NEB.com website is not compatible with the software version I am using?
15. Do you have Sample Sheets for use with the NEBNext Multiplex Small RNA Library Prep for Illumina?
16. Are NEBNext adaptor and primers for Illumina compatible with NEBNext reagents for Illumina library preparation?
17. Do I have to use custom sequencing primers for my libraries generated with NEBNext^® Small RNA Library prep kit for Illumina?

NEBNext Product Citations Search Tool

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Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

• E7300S_L_v1

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

• E7300G_v0_10013644
• E7300L_v0_10014027
• E7300S_v0_10011555
• E7300G_v0_10014242
• E7300S_v0_10013816
• E7300L_v0_10015547
• E7300G_v0_10016682
• E7300S_v0_10018271
• E7300L_v0_10021526
• E7300S_v0_10021655
• E7300L_v0_10027948
• E7300S_v0_10029243
• E7300G_v0_10031896
• E7300L_v0_10032502
• E7300L_v0_10034014
• E7300S_v0_10033595
• E7300S_v0_10035291
• E7300G_v0_10036038
• E7300S_v0_10037273
• E7300L_v0_10029841
• E7300S_v1_10041222
• E7300G_v0_10044476
• E7300S_v1_10045135
• E7300G_v0_10047239
• E7300S_v1_10029825
• E7300L_v1_10048038
• E7300S_v1_10048841
• E7300L_v1_10049566
• E7300S_v1_10051769
• E7300G_v0_10052875
• E7300L_v1_10037649
• E7300S_v1_10056660
• E7300L_v1_10057610
• E7300G_v0_10057655
• E7300S_v1_10037573
• E7300G_v0_10059451
• E7300L_v1_10060471
• E7300S_v1_10060462
• E7300G_v0_10061319
• E7300L_v1_10052980
• E7300S_v1_10062008
• E7300G_v0_10064721
• E7300L_v1_10065885
• E7300S_v1_10065066
• E7300L_v1_10073771
• E7300L_v1_10075523
• E7300S_v1_10072990
• E7300G_v0_10076196
• E7300S_v1_10052927
• E7300S_v1_10084715
• E7300S_v1_10088185
• E7300L_v1_10091054
• E7300G_v0_10093527
• E7300S_v1_10093071
• E7300S_v1_10066015
• E7300L_v1_10099790
• E7300L_v1_10101562
• E7300L_v1_10105355
• E7300S_v1_10105276
• E7300G_v0_10108057
• E7300S_v1_10109263
• E7300L_v1_10110351
• E7300G_v0_10111748
• E7300G_v0_10112748
• E7300S_v1_10112518
• E7300L_v1_10093650
• E7300S_v1_10119170
• E7300L_v1_10122933
• E7300G_v0_10093600
• E7300S_v1_10093623
• E7300S_v1_10127264
• E7300L_v1_10113290
• E7300G_v0_10135901
• E7300S_v1_10135703
• E7300S_v1_10113275
• E7300G_v0_10143242
• E7300L_v1_10150195
• E7300G_v0_10151643
• E7300S_v1_10150203
• E7300S_v1_10156114
• E7300G_v0_10113248
• E7300L_v1_10158167
• E7300S_v1_10163799
• E7300L_v1_10166864
• E7300S_v1_10169125
• E7300G_v0_10171431
• E7300S_v1_10174225
• E7300L_v1_10175482
• E7300S_v1_10136633
• E7300G_v0_10179136
• E7300G_v0_10180480
• E7300L_v1_10181897
• E7300S_v1_10181558
• E7300S_v1_10186127
• E7300G_v0_10172631
• E7300S_v1_10190701
• E7300S_v1_10172666
• E7300L_v1_10205709
• E7300S_v1_10208972
• E7300L_v1_10172649
• E7300G_v0_10219988
• E7300S_v1_10221932
• E7300L_v1_10193891
• E7300S_v1_10193905
• E7300S_v1_10236124
• E7300L_v1_10224170
• E7300S_v1_10224180
• E7300L_v1_10246272
• E7300S_v1_10258268
• E7300S_v1_10246281

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

• NEBNext 3´ Ligation Reaction Buffer
• Murine RNase Inhibitor
• NEBNext 5´ Ligation Reaction Buffer
• NEBNext 5´ Ligation Enzyme Mix
• LongAmp™ Taq 2X Master Mix
• Quick-Load pBR322 DNA-MspI Digest
• DNA Gel Elution Buffer
• Linear Acrylamide
• Nuclease-free Water
• TE Buffer
• NEBNext Index 1 Primer for Illumina
• NEBNext Index 2 Primer for Illumina
• NEBNext Index 3 Primer for Illumina
• NEBNext Index 4 Primer for Illumina
• NEBNext Index 5 Primer for Illumina
• NEBNext Index 6 Primer for Illumina
• NEBNext Index 7 Primer for Illumina
• NEBNext Index 8 Primer for Illumina
• NEBNext Index 9 Primer for Illumina
• NEBNext Index 10 Primer for Illumina
• NEBNext Index 11 Primer for Illumina
• NEBNext Index 12 Primer for Illumina
• Gel Loading Dye, Blue
• NEBNext 3´ SR Adaptor for Illumina
• NEBNext SR RT Primer for Illumina
• NEBNext SR Primer for Illumina
• NEBNext 5´ SR Adaptor for Illumina
• NEBNext First Strand Synthesis Reaction Buffer
• ProtoScript II Reverse Transcriptase
• NEBNext 3´ Ligation Enzyme Mix

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Trademarks

NEW ENGLAND BIOLABS®, LongAmp®, NEBNext® and QUICK-LOAD® are registered trademarks of New England Biolabs, Inc.

AGENCOURT® and AMPure® are registered trademarks of Beckman Coulter, Inc.
Agilent® and Bioanalyzer® are registered trademarks of Agilent Technologies, Inc.
Illumina® is a registered trademark of Illumina, Inc.
CORNING®, COSTAR® and SPIN-X® are registered trademarks of Corning, Inc.
NOVEX®, FIRSTCHOICE® and SYBR® are registered trademarks of Life Technologies, Inc.
FLASHPAGE™ is a registered trademark of Life Technologies, Inc.
Pellet Pestle® is a registered trademark of Kimble Kontes Asset Management, Inc.
Qiagen® and QIAQUICK® are registered trademarks of The Qiagen Group.