Genomic annotation files preparation

Required software:

1. Linux operating system (any version)

2. gtfToGenePred from UCSC tools

3. R programming language >=3.0

   
   

Two genomic annotations are used:

1. Download the mm10 genome fasta file here:

   wget //ftp.ensembl.org/pub/release-/fasta/mus_musculus/dna/Mus_musculus.GRCm38.dna.primary_assembly.fa.gz
   

   gunzip  Mus_musculus.GRCm38.dna.primary_assembly.fa.gz
   

2. Add the Synthetic Dux and tdTomato sequences to the downloaded mm10 fata. 

   wget https://raw.githubusercontent.com/sirusb/2CLike_analysis/master/Dux_tdTomato.fa
   

   cat Dux_tdTomato.fa >> Mus_musculus.GRCm38.dna.primary_assembly.fa
   

3. The standard mm10 gene annotation. It can be download from here (we used version GRCm38.85, but any newer version is also fine).

   wget ftp://ftp.ensembl.org/pub/release-85/gtf/mus_musculus/Mus_musculus.GRCm38.85.gtf.gz

   gunzip Mus_musculus.GRCm38.85.gtf.gz

4. Add the Synthetic Dux and tdTomato gtf annotations (see attachments) to the GTF file.

   wget https://raw.githubusercontent.com/sirusb/2CLike_analysis/master/SynDux_TdTomato.gtf

   cat Mus_musculus.GRCm38.85.gtf SynDux_TdTomato.gtf >> GRCm38.85_SynDux_TdTomato.gtf

5. Convert gtf to refFlat format using the gtfToGenePred tool.

   wget http://hgdownload.cse.ucsc.edu/admin/exe/linux.x86_64/gtfToGenePred

   gtfToGenePred GRCm38.85_SynDux_TdTomato.gtf GRCm38.85_SynDux_TdTomato.refFlat

6. the refFlat file need to be fixed in the R programming language as follows to be able to work with Drop-seq tools.

   refFlat <- read.table("GRCm38.85_SynDux_TdTomato.refFlat")

   options(scipen=999)

   refFlat$V11 <- refFlat$V1

   refFlat <- refFlat[,c(1,11,2:10)]

   toUse <- grep("chrNT", refFlat$V2,invert=T)

   refFlat = refFlat[toUse,]

   write.table(refFlat, file="GRCm38.85_SynDux_TdTomato.refFlat", sep="\t",col.names=F, row.names=F, quote=F)
   

7. The pseudo-genome that contains the sequences of repeats is manually created following the steps described in details in this bio-protocol page.
   

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