I am a newfie in this field. To practice before real experiment, I read the nature protocol (DOI: 10.1038/nprot.2017.016) and downloaded python code. To get the test data, I downloaded the fastq file () from the published paper (DOI: 10.1016/j.stem.2018.09.003).
Here, I performed the following code: “ python ./countspacers...
Black Incubus
over 3 years ago
Tapestation shows additional bands
I have been working according to the Joung et al 2017 paper and wanted to ask whether anyone has gotten additional unrelated bands on tapestation?
I pooled the pcr reactions from my amplified library and purified them on Zymo columns. Then I ran 2 micrograms from each sample on an agarose gel at low voltage so as not to he...
Can I demultiplex sequenced data based on indexes on the forward primers if doing single end sequencing on HiSeq?
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I have prepared amplicon libraries using a different combination of forward and reverse primers. The forward and reverse primers used had different indexes, and I am planning to do 100bp single-end se...
Lavender Gnome
almost 5 years ago
NGS 0 Match
I performed a HiSeq on my amplified lenti library and some transduced cells to determine representation and I'm getting 0 matches.
I checked for sequence correspondence with the published sgRNA library with no success, suggesting that either the amplification or NGS were incorrect and not just a misalignment.
Amplificatio...
Navy Dhampir
about 5 years ago
Troubleshooting Gecko Sequencing on HiSeq 4000
Has anyone used a HiSeq 4000 to sequence Gecko libraries? What % PhiX did you use? -we will have 6 different barcodes. Any other tips? or things to consider?
Thank you very much for your recent protocol paper for CRISPR screening. I have couple of questions about the NGS primers for amplifying the sgRNA library.
1) We are planning to deep-sequence both mouse and human GECKO libraries (2-vector system) to check sgRNA representations in the same Miseq run via multiplexing each l...
Purple Gremlin
over 6 years ago
Illumina Sequencing of GeCKO Library
I wanted to ask for some clarification on the Illumina primers used for sequencing before I actually go ahead and perform the run on the NextSeq machine.
1\. I cannot find the the indexes used in your excel guide (Readout primers for Illumina sequencing of sgRNA cassette : MS Excel \- ) anywhere in Illumina document of ind...
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