Single-cell RNA (ribonucleic acid) reverse transcription and library construction method

A reverse transcription and single-cell technology, applied in the field of transcriptome analysis, can solve the problems of inefficient transcription of sequences, biased transcript length, and low amplification efficiency, so as to avoid 3' bias, high efficiency and transcript amplification , the effect of less sample input

Active Publication Date: 2018-06-01
XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] 1. Not chain-specific
[0018] 2. Transcript length bias, unable to efficiently transcribe sequences exceeding 4Kb
[0020] 4.

Method used

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  • Single-cell RNA (ribonucleic acid) reverse transcription and library construction method
  • Single-cell RNA (ribonucleic acid) reverse transcription and library construction method
  • Single-cell RNA (ribonucleic acid) reverse transcription and library construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Obtaining single cell (or trace cell) total RNA

[0079] 1. Cell dilution: Dilute the mouse brain cells with 1x PBS buffer (the following PBS refers to 1×PBS buffer), centrifuge, resuspend, and process into a cell suspension with a density of ~20 cells / μL;

[0080] a. Tissue processing method: Grind the tissue in liquid nitrogen, add about 600 μL PBS for every 20-30 mg of tissue, centrifuge at 12,000 rpm (~13,400×g) for 1 minute to obtain a cell pellet, discard the supernatant and aspirate the cell pellet, and use Dilute 1x PBS into cell suspension;

[0081] b. Monolayer culture cell treatment method: Blow down the adherent cells gently with a P1000 pipette, centrifuge at 12,000rpm (~13,400×g) for 1 minute to obtain a cell pellet, discard the supernatant and aspirate the cell pellet, wash with 1x PBS Diluted into cell suspension;

[0082] c. Cell suspension treatment method: Centrifuge at 12,000rpm (~13,400×g) for 1 minute to obtain cell pellets, discard the...

Embodiment 2

[0089] Example 2 reverse transcription to obtain double-stranded full-length cDNA

[0090] 1. Add 1.7*NμL of RT Enzyme Mix to the RT Buffer in the previous step, mix manually, and centrifuge;

[0091] 2. Add 15 μL of the mixture of RT Buffer and RT Enzyme Mix to each cell lysate, put it on ice immediately after centrifuging briefly, and incubate the sample on a preheated PCR machine, the conditions are as follows:

[0092]

[0093] Example 3 Amplification

[0094] Add 29.25 μL of PCR Mix to the reverse transcription product (the solution volume is 49.25 μL at this time), add 0.75 μL of Index primers to each reaction tube, mix well and centrifuge, and amplify in a PCR instrument. The reaction conditions are as follows:

[0095]

[0096]

Embodiment 4

[0097] Example 4 Library detection

[0098] Step 1: Purification

[0099] 1) Transfer the amplified product to a centrifuge tube, take 0.8× (40uL) Ampure XP magnetic beads or CMpure magnetic beads, mix with the amplified product and place it on a magnetic stand for 10 minutes;

[0100] 2) After the magnetic beads are completely adsorbed to the tube wall (about 5 min), discard the supernatant, wash the magnetic beads twice with freshly prepared 80% ethanol, and discard the supernatant;

[0101] 3) Let stand at room temperature for 5 minutes, and after the magnetic beads are dry (please be careful not to crack the magnetic beads due to excessive drying, so as not to affect the recovery efficiency), resuspend the magnetic beads with 17.5uL TE buffer, EB buffer or nucleic acid-free water according to downstream needs;

[0102] 4) After standing at room temperature for 5 minutes, place the centrifuge tube on a magnetic stand, and absorb 15uL of supernatant, which is a double-stran...

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Abstract

The invention belongs to the field of transcriptome analysis and relates to a quick single-cell RNA (ribonucleic acid) reverse transcription and library construction method. According to the method, 20-500ng high-quality full-length double-chain cDNA (complementary deoxyribonucleic acid) is amplified by taking 1-2000 cells or 10pg-20ng extracted eukaryote total RNA as an initial, and a high-quality cDNA library meeting downstream analysis requirements is obtained. The method effectively avoids 3' preference and genome DNA contamination in a cDNA synthesis process; an expression quantitation molecule label can assist in gene expression quantity calculation; and at the same time, the expression quantitation molecule label can also keep chain source information during complete amplification of RNA sequence information. The method can achieve a reverse transcription and amplification library construction success rate of above 95%; the cDNA library can be connected with an Illumina main stream sequencing platform; lower computer data (5M Reads) can detect gene expression of above 90%; the gene expression consistency exceeds 90%; amplification has no obvious bias; and a required sample input amount is smaller.

Description

technical field [0001] The invention belongs to the field of transcriptome analysis and relates to a rapid single-cell RNA reverse transcription and library construction method. Background technique [0002] Transcriptome refers to the collection of all transcribed mRNA products in a certain species or a specific cell in a certain physiological function state, including time and space limitations, and is an inevitable link connecting the genetic information of the genome with the proteome of biological functions . [0003] Transcriptome analysis includes but is not limited to: analysis of coding genes, prediction of translated proteins, splicing of exons and introns, structural analysis and functional analysis of transcripts, secondary structure of mRNA, differential expression of genes, etc. At present, mature and reliable transcriptome analysis methods include RT-qPCR analysis of gene expression, microarray hybridization platform analysis and analysis of transcriptional a...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6869C40B50/06
CPCC12N15/1096C12Q1/6806C12Q1/6869C40B50/06C12Q2521/107C12Q2525/191C12Q2531/113C12Q2535/122
Inventor 胡春旭陆思嘉任军
Owner XUKANG MEDICAL SCI & TECH (SUZHOU) CO LTD
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