Whole transcriptome amplification of the mRNA was done using the Smart-seq protocol (Ramsköld et al., 2012 (link)). Briefly, Agencourt RNAClean beads were used to isolate the mRNA, followed by reverse transcription reaction using SMARTScribe (Clontech) enzyme. cDNA was isolated using Agencourt AMPure XP beads, followed by PCR amplification using the Advantage2 kit (Clontech). After another wash with the AMPure beads, DNA quality and concentration were measured on randomly selected wells using High Sensitivity DNA Chips for Bioanalyzer (Agilent technologies). This information was used to estimate DNA concentrations in the other wells, and to eliminate empty wells. Cells were labeled and sequencing libraries were prepared using Nextera XT DNA library preparation kit (Illumina), after wells were diluted in TE according to their estimated concentration. After all wells from a given plate were pooled and washed using AMPure XP beads, samples were sequenced using Rapid flowcell on an Illumina HiSeq2500 sequencer.
Ampure xp beads
Manufactured by Takara Bio
AMPure XP beads are magnetic beads used for the purification of nucleic acids, including DNA and RNA, in various molecular biology applications. The beads selectively bind to nucleic acids and allow for the removal of contaminants and unwanted materials through a simple washing process. AMPure XP beads provide a reliable and efficient way to purify nucleic acids from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500 sequencer, by Illumina (1 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
High sensitivity dna chips for bioanalyzer, by Agilent Technologies (1 mentions)
Smartscribe, by Takara Bio (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Truseq nano dna kit, by Illumina (1 mentions)
Ampure xp spri beads, by Beckman Coulter (1 mentions)
Advantage 2 polymerase mix, by Takara Bio (1 mentions)
Smarter ultra low rna kit for sequencing, by Illumina (1 mentions)
Nextera xt dna, by Illumina (1 mentions)
2 protocols using ampure xp beads
1
Whole Transcriptome Amplification using Smart-seq
2
Single-Cell RNA-Seq Library Preparation
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cDNA synthesis was performed using the SMARTer Ultra Low RNA kit for Illumina sequencing (Clontech) according to the manufacturer’s recommendations. Then 3000–5000 sorted GFP+ cells were directly lyzed in 3.5 μl of reaction buffer and immediately frozen at –80 °C. cDNAs were synthesized, purified with Ampure XP beads and then amplified with 13 PCR cycles with Advantage 2 Polymerase Mix (50×, Clontech). The PCR products were purified on SPRI Ampure XP beads (Beckman Coulter), and the size distribution was checked on a high-sensitivity DNA chip (Agilent Bioanalyzer). cDNA libraries were prepared with TruSeq Nano DNA kit or Nextera XT DNA (Illumina). For TruSeq Nano libraries, 20–30 ng cDNA was sheared by sonication (parameters adjusted to obtain fragments from 350 to 450 bp). For Nextera libraries, 1 ng was fragmented by tagmentation. Then cDNA libraries were prepared according to the manufacturer’s recommendations. Samples were sequenced on an Illumina HiSeq 2000 at an average of 72.3 million 100-bp paired-end reads. RNA-seq data have been deposited in the European Nucleotide Archive from EMBL-EBI [38 ].
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