Total RNA was purified using a miRNAeasy micro kit (Qiagen, USA) and quantified as described previously (12 (link), 15 (link)). Purified total RNA (5 ng) was amplified following the Smart-seq2 protocol (16 (link)). cDNA was purified using AMPure XP beads (1:1 ratio, Beckman Coulter). From this step, 1 ng of cDNA was used to prepare a standard Nextera XT sequencing library (Nextera XT DNA sample preparation kit and index kit, Illumina). Samples were sequenced using a HiSeq2500 (Illumina) to obtain 50-bp single end reads. Both whole-transcriptome amplification and sequencing library preparations were performed in a 96-well format to reduce assay-to-assay variability. Quality control steps were included to determine total RNA quality and quantity, the optimal number of PCR pre-amplification cycles, and fragment size selection. Samples that failed quality control were eliminated from further downstream steps. Barcoded Illumina sequencing libraries (Nextera, Illumina) were generated utilizing the automated platform (Biomek FXp). Libraries were sequenced on the HiSeq2500 Illumina platform to obtain 50-bp single end reads (TruSeq® Rapid Kit, Illumina), generating a total of >700 million mapped reads (median of ~9 million mapped reads per sample).
Biomek fxp
Manufactured by Illumina
The Biomek FXp is a liquid handling workstation designed for automated liquid transfers and sample preparation. It features a modular design and versatile pipetting capabilities to support a variety of laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Ampure xp bead, by Beckman Coulter (3 mentions)
Truseq rapid kit, by Illumina (2 mentions)
Nextera xt dna sample preparation kit and index kit, by Illumina (2 mentions)
Mirnaeasy micro kit, by Qiagen (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Elution buffer, by Qiagen (1 mentions)
Nextera xt library preparation kit, by Illumina (1 mentions)
Hiseq 4000 sequencer, by Illumina (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
High sensitivity dna chip, by Agilent Technologies (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
3 protocols using biomek fxp
1
Single-cell RNA-seq Library Preparation and Sequencing
2
Smart-seq2 and Bulk RNA-seq Library Prep
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell RNA-seq libraries were prepared as per the Smart-seq2 protocol by Picelli et al.17 (link) with minor technical adaptations. Reverse transcription was carried on with 0.25 U/reaction of SuperScript II (Invitrogen) and PCR pre-amplification with 5′ biotinylated IS PCR primers (Biomers) for 22 cycles. PCR cleaning was performed by using Ampure XP Beads (Beckman Coulter) at a ratio 0.8:1 (beads:cDNA). cDNA was re-suspended in elution buffer (Qiagen) and assessed with a High-sensitivity DNA chip (Agilent).
For Bulk RNA-seq RNA was extracted from 5000 sorted cells by using the Qiagen RNeasy Micro Kit according to the manufacturer’s instructions. One microliter of RNA was added to the respective wells in the plate containing lysis buffer and processed as for single cells with the exception of the PCR pre-amplification step that was carried out for 18 cycles. Barcoded Illumina sequencing libraries (Nextera XT, Library preparation kit, Illumina) were generated using the automated platform (Biomek FXp). Finally, libraries were pooled, and sequencing was performed in paired-end mode for 2×75 bp cycles using the Illumina HiSeq 4000 sequencer.
For Bulk RNA-seq RNA was extracted from 5000 sorted cells by using the Qiagen RNeasy Micro Kit according to the manufacturer’s instructions. One microliter of RNA was added to the respective wells in the plate containing lysis buffer and processed as for single cells with the exception of the PCR pre-amplification step that was carried out for 18 cycles. Barcoded Illumina sequencing libraries (Nextera XT, Library preparation kit, Illumina) were generated using the automated platform (Biomek FXp). Finally, libraries were pooled, and sequencing was performed in paired-end mode for 2×75 bp cycles using the Illumina HiSeq 4000 sequencer.
3
Streamlined RNA-seq Library Preparation
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!