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how many reads per sample would you recommend in single cell RNA-Seq (e.g. Cel-Seq) super0925 RNA Sequencing 0 09-06-2016 02:02 PM

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Old 12-09-2016, 04:00 AM   #1
StephanK
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Location: Europe

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Default Cel Seq2 Exonuclease and cell barcodes

Hi!

I aim to start a Cel Seq2 experiment to get transcriptome data from single cells. The paper is here: https://genomebiology.biomedcentral....059-016-0938-8

There are a few unclear things to me.

-Why is there an ExoSAP IT treatment? I understand it is being used to remove primers but thats also what the SPRI beads are doing, or?

-According to the protocol, the ExoSAP IT treatment is after the IVT reaction, in contrast to the Cel Seq/MARS seq procedure. Does that not leave many T7 primers in the cDNA mix that would decoy the polymerase?

-Would you recommend pooling 96 samples and run that on one lane? The read starts with the UMI /(6bp) and then enters the cell barcode. Should that provide enough complexity for the Illumina 2500 HiSeq? Our seq facility is unsure about that and they mentioned they only use barcodes with a hamming distance of at least 5, the ones in the paper should have at least 2. Especially, what if I mix two plates and distinguish them with the Illumina RPIndex Primers?

Thank you and all the best,
Stephan
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