Cell suspensions was generated by washing melanoma cells from different cultures with PBS followed by trypsinizing with TripLE Xpress (Invitrogen, Darmstadt, Germany) for 5 minutes. The reaction was stopped by adding media containing FCS. Single melanoma cells from short-term cultures were captured on an integrated fluidic circuit RNA-seq chip (Fluidigm, Hamburg, Germany) using the Fluidigm C1 system. Cells were loaded onto the chip at a concentration of 200 cells/μL and imaged by phase-contrast. Cell capture, cell lysis, reverse transcription, and cDNA amplification were performed on the chip as described [50 (link)]. Illumina libraries were constructed using the Illumina Nextera XT DNA Sample Preparation kit using the protocol supplied by Fluidigm. Sequencing libraries were pooled (3 μL each) and purified with 18% SPRI beads. Library concentration and size distribution were assessed on an Agilent Bioanalyzer and with Qubit dsDNA HS Assay kits and a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Invitrogen, Darmstadt). Each cell was paired-end sequenced (100 base reads) on an Illumina HiSeq 2500 to a depth of 2–5 million reads and base-calling, adaptor trimming, and de-multiplexing were performed as described [51 (link), 52 (link)].
Nextera xt dna sample preparation kit
Manufactured by Standard BioTools
The Nextera XT DNA Sample Preparation kit is a laboratory equipment product from Standard BioTools. It is designed for library preparation of DNA samples prior to sequencing. The kit provides reagents and protocols for fragmentation, tagging, and amplification of DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
C1 platform, by Standard BioTools (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Triplexpress, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Agilent dna high sensitivity chip, by Agilent Technologies (1 mentions)
Smarter ultra low rna kit for, by Illumina (1 mentions)
Qubit high sensitivity dna kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500 or 4000, by Illumina (1 mentions)
Smarter ultra low rna kit, by Takara Bio (1 mentions)
Facs tube, by BD (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Smarter ultra low rna kit, by Illumina (1 mentions)
Hiseq 2500 machine, by Illumina (1 mentions)
4 protocols using nextera xt dna sample preparation kit
1
Single-Cell RNA-Seq of Cultured Melanoma Cells
2
Single-cell RNA-seq of Aged Mouse HSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lin−Sca-1+c-Kit+CD150+CD48− cells (2,000) from young and old Vwf-EGFP mice were sorted in 2 μl filter-sterilized PBS with 50% FCS (ref. 12 (link)). Single cells were captured on a small-sized (5–10 μm cell diameter) C1 Single-Cell Auto Prep IFC for messenger RNA Sequencing (Fluidigm) using the Fluidigm C1 system. Cells were loaded onto the chip at a concentration of ∼400 cells μl−1 and imaged by phase-contrast microscopy to check single cell per capture site. Sixty-one young HSCs and 74-old HSCs were captured and used for RNA-seq. Cells were lysed and whole-transcriptome amplified cDNA prepared on the C1 Fluidigm chip according to the manufacturer's protocol, using SMARTer Ultra Low RNA kit for Illumina (Clontech). Single-cell cDNA libraries were quantitated by an Agilent Bioanalyzer using High-Sensitivity DNA chip. Illumina libraries were constructed in 96-well plates using the Illumina Nextera XT DNA Sample Preparation kit according to a protocol supplied by Fluidigm. Indexed libraries size and quality was checked using Agilent High-Sensitivity DNA chip. The concentration of indexed libraries was determined using Qubit High-Sensitivity DNA kit (Invitrogen). Libraries were pooled to a final concentration of 4–5 nM and were sequenced on an Illumina HiSeq 2,000 (single end 51 base pair reads) at SciLifeLab, Karolinska Institutet, Stockholm, Sweden.
3
Single-Cell RNA Sequencing of Dissociated NPCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NPs were dissociated using 0.05% Trypsin (Gibco) spun down in ES-medium, resuspended, washed, and spun down in 10 ml PBS (Gibco). Afterwards, cells were resuspended in 1 ml N2B27 and filtered into a FACS tube (Falcon). The Fluidigm C1 platform was used to capture individual cells using 96 small or medium IFC chips. Cells were diluted in the range of 250,000–400,000 cells per ml for chip loading. Capturing efficiency was evaluated by manually inspecting each capture site on the chip using the automated NanoEntek JuLi cell imager. Only capture sites containing single cells were processed for library preparation and sequencing. Single cell full-length cDNA was generated using the Clontech SMARTer Ultra Low RNA kit on the C1 chip using manufacturer-provided protocol. ArrayControl RNA Spikes (AM1780) were added to the cell lysis mix, as recommended in the Fluidigm protocol. Libraries were prepared using the Illumina Nextera XT DNA Sample Preparation kit, according to a protocol supplied by Fluidigm, and sequenced on Illumina Hiseq 2500 or 4000 using 50- or 75-bp paired-end runs.
4
Single-Cell RNA-seq of Activated T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells from the two donors (#42 and #123, from a previous study [3 (link)]) were TCR-activated as previously described, and maintained in 2 ml OpTmizer expansion medium with 5% FBS and100 U/ml IL-2 for 72h. Dead cells were removed by centrifugation on a 3 ml Percoll gradient at 800 x g for 10 minutes. Cells were washed, counted and resuspended in PBS. Cells were either used for bulk RNA-Seq or loaded to the Fluidigm C1 platform for single-cell capture and single-cell RNA-seq library generation according to the manufacturer’s protocol. Briefly, a cell suspension of approximately 300,000 cells/ml was introduced into the medium size chip (10–17 μm plate), suitable to capture cells of 10–17 ± 2 μm, and thus able to capture activated cells (usually ranging from 10 to 13 μm). After cell separation and capture, empty or debris-occupied wells were identified by microscope visualisation and discarded from subsequent analysis. cDNA libraries were then produced directly and automatically on the chip with Clontech SMARTer Ultra Low RNA kit for Illumina using manufacturer-provided protocols. Illumina libraries were constructed in 96-well plates using the Illumina Nextera XT DNA Sample Preparation kit according to a protocol supplied by Fluidigm and sequenced on HiSeq2500 machine (Illumina), with 50 bp paired-end, 14 libraries multiplexed per lane.
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!