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Old 02-24-2016, 08:29 AM   #8
Location: Germany

Join Date: Jan 2013
Posts: 17

@ SylvainL:

The paper "Revisiting Global Gene Expression Analysis" was thought to give people an idea of the problem i'm facing. I don't think it makes sense to discuss the quality here.
To give everybody an ide of the problem without checking the paper, the pic:

1st row: for a transcription factor regulating only a few genes, no spike ins are required for sure. Normalization works perfect.
2nd row: if a transcription factor changes most of the genes (i've heard alredy 20 % of all genes is enough), the normalization will be biased, because the normlization programs assume that the expression of most genes will stay the same.
3rd row: with the ERCCs included, the normalization bias mentioned in row 2 can be avoided. That's what i want to use the spike ins for.

My protein is a transcriptional activator in a viral system, but in the human system it downregulated most of the genes. This was kind of unexpected. Therefore i want to exclude that i get the normalization bias, which is described in the picture.

@ Thank you, i can also use bowtie 2, nevertheless i already made a file which includes each ERCCs RNA like a single chromosome.

Maybe someone can nevertheless tell me, how to do the normalization. I will for sure check both ways of analysis (with and without ERCCs), but therefore i would need to know how to normalize with the ERCCs.
Thanks a lot
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