A method for single-cell RNA reverse transcription and library construction

A reverse transcription, single-cell technology, applied in the field of transcriptome analysis, can solve the problems of transcript length bias, material loss, low amplification efficiency, etc., and achieve the effect of avoiding 3' bias and less sample input

Active Publication Date: 2021-05-28
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. Purification steps may result in loss of material
[0031] 1. Not chain-specific
[0032] 2. The amplification efficiency is not high

Method used

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  • A method for single-cell RNA reverse transcription and library construction
  • A method for single-cell RNA reverse transcription and library construction
  • A method for single-cell RNA reverse transcription and library construction

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 method for rapidly performing single-cell RNA reverse transcription and library construction. The invention can start from 1-2000 cells or 10pg-20ng of extracted eukaryotic total RNA within 5-6 hours, amplify 20-500ng high-quality full-length double-stranded cDNA, and obtain downstream High-quality cDNA libraries required for analysis. This invention effectively avoids 3' bias and genomic DNA contamination during cDNA synthesis. Expression quantitative molecular tags can assist in the calculation of gene expression. At the same time, the expression quantitative molecular tags can also retain strands while completely amplifying RNA sequence information. source information. The invention can obtain more than 95% success rate of reverse transcription and amplification library construction, the cDNA library can be seamlessly connected to the mainstream sequencing platform of Illumina, and the off-machine data (5M Reads) can detect more than 90% gene expression, and the gene expression consistency More than 90%, the amplification has no obvious bias, and the required sample input is less.

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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Patent Type & Authority Patents(China)
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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