Hi all,

I have an RNAseq experiment confounded by batch effect. Its a time-course design with the following setup:

    time batch
1     0     1
2     0     1
3   1.5     1
4   1.5     1
5   1.5     1
6     3     1
7     3     1
8     3     1
9    3D     2
10   3D     2
11   3D     2
12   12     1
13   12     1
14   12     1
15   24     1
16   24     1
17   24     1
18   24     1
19  24c     1
20  24c     1

I need to compare 3vs0 and 3Dvs0, but 3 and 3D are two different treatments at the same time point, and they are in different batches, so its impossible to separate biological effect from the possible batch effect.

However, I can define the negative control genes - genes which are not affected by two types of treatments, but probably are affected by the batch effect. So my idea is use RUVg package, which in theory can infer the batch effect based on these negative controls, and account for it in down-stream DESeq2analysis.

I'd appreciate if anyone can comment whether this approach is reasonable or not and I if anyone had a similar situation before. Thank you very much in advance