Each EEC or PEC sample obtained after FACS was directly pelleted by centrifugation and resuspended in 3.5 μl of reaction buffer, lysed by freezing in liquid nitrogen and stored at − 80 °C according to the Smart-seq2 protocol [63 (link)]. cDNA was synthesized and amplified by a 13-cycle PCR reaction. The quality of cDNA was verified by 2100 High Sensitivity DNA assay (Agilent Technologies); 1-ng cDNA was used for preparing each cDNA library using the Nextera-XT kit (Illumina) and sequenced on Hi-seq 2000 to obtain around 40–60 millions of reads (100 base paired-ends).
2100 high sensitivity dna assay
Manufactured by Agilent Technologies
The 2100 High Sensitivity DNA assay is a laboratory instrument designed to analyze and quantify low concentrations of DNA samples. It provides accurate and sensitive measurements of DNA quantities, enabling researchers to work with limited sample materials. The core function of this assay is to deliver reliable DNA analysis data to support various scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt kit, by Illumina (2 mentions)
Nextera xt dna kit, by Illumina (1 mentions)
Nextseq 500 sequencer, by Illumina (1 mentions)
Smarter ultra low input rna kit, by Takara Bio (1 mentions)
Agilent rna 6000 pico chip, by Agilent Technologies (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
Hiseq 2000 sequencer, by Illumina (1 mentions)
4 protocols using 2100 high sensitivity dna assay
1
Single-Cell RNA-seq Library Preparation
2
Single-cell RNA-seq library preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each FACS-sorted cells sample was directly pelleted by centrifugation and resuspended in 3.5 μl of reaction buffer, lysed by freezing in liquid nitrogen and stored at -80°C according the Smart-seq2 protocol [47 (link)]. cDNA was synthesised and amplified by a 13 cycles PCR reaction. Quality of cDNA was verified by 2100 High Sensitivity DNA assay (Agilent technologies). 1 ng cDNA was used for preparing each cDNA library using Nextera-XT kit (Illumina) and sequenced on Hi-seq 2000 to obtain around 20 millions of reads (75 base single-end).
3
Single-cell RNA-seq protocol for zebrafish
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
cDNA was obtained using the SMART-seq2 protocol as recently described48 (link). Briefly, each cell preparation was pelleted and lysed in Lysis Buffer by freezing in liquid nitrogen and stored at − 80 °C. Synthesis of cDNA was performed directly on the lysed cells; cDNA were amplified by 10 PCR cycles and purified before assessing their quality on the bioanalyzer (2100 high sensitivity DNA assay, Agilent Technologies). One hundred and fifty pg of cDNA were used as input to prepare the libraries using the Nextera XT DNA kit (Illumina). Seventy five bp single-end sequences were obtained using the NextSeq500 Illumina Sequencer with coverage of about 20 million reads per library.
Raw reads were aligned to the zebrafish genome (Zv9, Ensembl genome version 79, ensembl.org) using STAR software49 (link). Normalization and differential expression analysis were performed using DESeq250 (link). Genes were considered differentially expressed with FDR < 0.01 (False Discovery Rate).
Raw reads were aligned to the zebrafish genome (Zv9, Ensembl genome version 79, ensembl.org) using STAR software49 (link). Normalization and differential expression analysis were performed using DESeq250 (link). Genes were considered differentially expressed with FDR < 0.01 (False Discovery Rate).
4
RNA Extraction and Sequencing from FACS Sorted Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from FACS sorted cells using the RNeasy plus micro kit (Qiagene). RNA from endocrine and acinar cells was eluted in 10 μL with a concentration of 100–400 pg/μL. RNA integrity was assessed by a capillary electrophoresis using Agilent RNA 6000 pico chip (Agilent technologies), the RIN value for each sample was from 8 to 10. The Smarter Ultra low RNA input kit (clontech) [82 (link)] was used to for the synthesis and amplification of cDNA synthesis using up to 10 ng of total RNA following the manufacturer’s instructions and performing no more than 12 cycles of PCR in order to minimize amplification biases. The quality of cDNA was verified by 2100 High Sensitivity DNA assay (Agilent technologies). Truseq DNA Illumina libraries were prepared and sequenced to obtain approximately 90 million reads (100 bp paired-end reads) per library using the Hiseq 2000 Illumina sequencer.
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!