50,000 – 100,000 hPSC-derived microglia from 3 different hPSC lines, H1, H9, and the wildtype iPSC line SA241–1, were sorted from neuronal co-cultures by CX3CR1+. RNA was extracted using the Zymo RNA Micro Kit. RNA from primary human microglia was obtained after sorting postmortem tissue from the frontal and temporal lobes from patients aged 60–77 years. All samples were submitted to the MSKCC Integrated Genomics Core for paired end SMARTER-sequencing and 30–40 million reads. Analysis was done through a standard pipeline through the MSKCC Bioinformatics core: FASTQ files were mapped using the rnaSTAR aligner. Output SAM files are processed using PICARD tools. The mapped reads were then processed using HTSeq to compute a raw expression count matrix, which was then processed using DESeq from R/BioConductor to analyze differential expression between samples.
Rna micro kit
Manufactured by Zymo Research
The RNA Micro Kit is a laboratory equipment designed for the extraction and purification of small amounts of RNA from various sample types. The kit utilizes a specialized membrane-based technology to efficiently capture and purify RNA, making it suitable for applications that require high-quality RNA from limited sample material.
Automatically generated - may contain errors
4 protocols using rna micro kit
1
Transcriptome Analysis of hPSC-Derived Microglia
2
Multi-Modal Single Cell Analysis Protocol
3
Transcriptome Analysis of hPSC-Derived Microglia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
50,000 – 100,000 hPSC-derived microglia from 3 different hPSC lines, H1, H9, and the wildtype iPSC line SA241–1, were sorted from neuronal co-cultures by CX3CR1+. RNA was extracted using the Zymo RNA Micro Kit. RNA from primary human microglia was obtained after sorting postmortem tissue from the frontal and temporal lobes from patients aged 60–77 years. All samples were submitted to the MSKCC Integrated Genomics Core for paired end SMARTER-sequencing and 30–40 million reads. Analysis was done through a standard pipeline through the MSKCC Bioinformatics core: FASTQ files were mapped using the rnaSTAR aligner. Output SAM files are processed using PICARD tools. The mapped reads were then processed using HTSeq to compute a raw expression count matrix, which was then processed using DESeq from R/BioConductor to analyze differential expression between samples.
4
Multi-Modal Single Cell Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For single molecule FISH, samples were run using the RNAscope 2.5 LS fluorescent multiplex assay (automated). Slides were imaged on the Perkin Elmer Opera Phenix High-Content Screening System, in confocal mode with 1 μm z-step size, using 20X (NA 0.16, 0.299 μm/pixel) and 40X (NA 1.1, 0.149 μm/pixel) water-immersion objectives.
For flow cytometry, mononuclear cells (MNCs) were either stained ex vivo or post activation with PMA+I for two hours. Cells were stained with the live/dead marker Zombie Aqua for 10 minutes at room temperature, and then washed with PBS+0.5%FCS, with the CD8 and B cell panels of antibodies.
qPCR was performed in three spleen samples. Cells were stained with the live/dead marker Zombie Aqua for 10 minutes at room temperature, and then washed with PBS+0.5%FCS, followed by staining with the antibodies at 4°C for 45 minutes. Cell sorting was performed on a BD Fusion 4 laser sorter and RNA was extracted using a Zymo Research RNA micro kit.
For immunofluorescence, samples were fixed in 1% paraformaldehyde for 24 hours followed by 8 hours in 30% sucrose in PBS, and were stained for 2h at RT with the appropriate antibodies, washed three times in PBS and mounted in Fluoromount-G® (Southern Biotech). Images were acquired using a TCS SP8 (Leica, Milton Keynes, UK) confocal microscope.
For flow cytometry, mononuclear cells (MNCs) were either stained ex vivo or post activation with PMA+I for two hours. Cells were stained with the live/dead marker Zombie Aqua for 10 minutes at room temperature, and then washed with PBS+0.5%FCS, with the CD8 and B cell panels of antibodies.
qPCR was performed in three spleen samples. Cells were stained with the live/dead marker Zombie Aqua for 10 minutes at room temperature, and then washed with PBS+0.5%FCS, followed by staining with the antibodies at 4°C for 45 minutes. Cell sorting was performed on a BD Fusion 4 laser sorter and RNA was extracted using a Zymo Research RNA micro kit.
For immunofluorescence, samples were fixed in 1% paraformaldehyde for 24 hours followed by 8 hours in 30% sucrose in PBS, and were stained for 2h at RT with the appropriate antibodies, washed three times in PBS and mounted in Fluoromount-G® (Southern Biotech). Images were acquired using a TCS SP8 (Leica, Milton Keynes, UK) confocal microscope.
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!