Anyone here knows how to add a new gene (GFP) to customer reference by using cellranger? We use the Cre/loxp system by using R26RmTmG mice and want to use GFP to isolate specific cells. I have read the cellranger workflow but still, have no idea how to do it. Like it is said that I should update the GTF file as shown in the screenshot.
Step 1: Add the the GFP sequence to your fasta file
Step 2: Annotate the gtf so that it contains all the necessary information to annotate your information
Add this line to your GTF file but modify where necessary: "Make sure that all columns are tab separated"
GFP_name AddedGene exon 1 1966 . + 1 gene_id "GFP"; transcript_id "GFP"; gene_name "GFP"; transcript_name "GFP"
- The first column has to correspond to the header of the fasta sequence
- start and end indicates the length of your sequence. I added a 1966 bp long sequence to my gtf
Code example: step 1:
cat reference.fa gfp_seq.fa > new_reference.fa
cat genes.gtf gfp.gtf > new_genes.gtf
- gfp_seq.fa is your custom gene in fasta file - gfp.gtf is your custom gtf line or lines if you want to add more
You can also open your current reference fasta and gtf file in a text editor and copy paste the sequence and gtf line in there.
cellranger mkref on your new_reference and new_genes file