YeaCell™ 1st Strand Synthesis kit for Single Cell 3ʹRNA-seq

Details:

Product description

YeaCellTM 1st Strand Synthesis kit for Single Cell 3ʹ RNA-seq can use Oligo (dT)18 or TSO Primer to reverse transcription of single cell experiment, which was suitable for microfluidic microsystems. The cDNA of the product can be enriched by PCR reaction with universal primers, and then constructed a DNA library using Yeasen's enzymatic library construction kit. This product uses a new reverse transcriptase based on M-MLV(RNase H-)Reverse Transcriptase through multi-point mutation, which has the advantages of high reverse transcriptase efficiency, low mismatch rate and high fidelity.

Specifications

Cat.No.

13594ES04 / 13594ES08 / 13594ES95

Size

4 T / 8 T / 120 T

Components

Components No.

Name

13594ES04

13594ES08

13594ES95

13594-A

RT buffer

76 μL

152 μL

2×1130 μL

13594-B

RT Enzyme mix

36 μL

72 μL

1056 μL

Storage

This product should be stored at -25~-15℃ for 1 years.

Application

1. High-throughput single-cell full-length cDNA synthesis for mammalian or eukaryotic cells without cell walls.
2. 10 pg~1 μg total RNA with poly (A).
3. This product is not suitable for prokaryotic total RNA and degraded RNA, such as FFPE RNA.

Instructions 

Plan A : If the experimental scheme is a high-throughput single-cell experiment, appropriate adjustments should be made according to different single-cell instruments.

  1. To prepare the Reaction Buffer, thaw the RT buffer, Template Switch Oligo (customer)and Reducing Agent B (customer) at room temperature in advance, mix it upside-down, centrifuge it at the bottom of the tube and place it on ice for use.

Table 1 Reaction Buffer prepare

Name

ValueμL

RT buffer

18.8

Template Switch Oligo

2.4

Reducing Agent B

2

RT Enzyme mix

8.8

Total

32

  1. Mix thoroughly by gently pipetting up and down at least 10 times. Briefly spin down the tube in a microcen- trifuge to collect the liquid from the side of the tube.
  2. Refer to the instructions for Cat#12520ES (for 10x Genomics single cell instrument) for subsequent use.

Plan B : For reverse transcription experiments, refer to this procedure

Step 1 RNA denaturation

  1. Prepare the reaction liquid according to Table 2:   

Table 2 RNA denaturation reaction

Name

ValueμL

Oligo (dT)1820~50 mM(customer)

1

Lysis cell or RNA

12

Total

13

  1. Mix thoroughly by gently pipetting up and down at least 10 times. Briefly spin down the tube in a microcen- trifuge to collect the liquid from the side of the tube.
  2. Place tube in a thermocycler and run the following program:heated lid 80℃ on70℃5 min,then place it on ice immediatly.

Step 2 1st Strand Synthesis

  1. Remove the first chain of synthetic reagents from -20°C, thaw at room temperature, mix thoroughly and spin down. Prepared the 1st strand synthesis reagents according to the Table 3.

Table 3 1st Strand Synthesis reaction

Name

ValueμL

Above step

13

5×RT Buffer

4

TSO Primer20~50 mM(customer)

1

RNase Inhibitor(40 U/µL)customer

1

Reverse Transcriptase for Single Cell Full Length cDNA

1

Total

20

  1. Mix thoroughly by gently pipetting up and down at least 10 times. Briefly spin down the tube in a microcen- trifuge to collect the liquid from the side of the tube.
  2. Place tube in a thermocycler and run the following program:heated lid 105℃ on4290 min7015min4℃hold
  3. The product can be directly used for two-strand cDNA synthesis or temporary storage at -80℃.

Notes

1. This product is for research use only.

2. Please operate with lab coats and disposable glovesfor your safety.

3.Supplies free of RNase contamination and cleaning the experimental area regularly are necessary. ThermoFisher's RNAZapTM high-efficiency nucleic acid removal spray was recommended to remove RNase contamination.

4. Other materials should be asked if we want to construct a DNA library.

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