Downlaod raw data for analysis

VAR=$(tail -n +2 SraRunTable.txt | cut -d ',' -f 1)

for i in ${VAR}
	do
				fastq-dump ${i}
	done

Run fastqc

Align to the genome

VAR=$(tail -n +2 ../meta_data/SraRunTable.txt | cut -d ',' -f 1)

for i in ${VAR}
	do
		
		bowtie2 -p 8 -q --local -x ../reference/mm9 -U ../raw_data/${i}.fastq -S ../results/bowtie2/${i}.sam
	done

Convert sam file to bam file

VAR=$(tail -n +2 ../meta_data/SraRunTable.txt | cut -d ',' -f 1)

for i in ${VAR}
	do
		
		samtools view -h -S -b ../results/bowtie2/${i}.sam -o ../results/bowtie2/${i}.bam
		#echo ${i}
	done

Sort bam file

VAR=$(tail -n +2 ../meta_data/SraRunTable.txt | cut -d ',' -f 1)

for i in ${VAR}
	do
		
		samtools sort -t 2 -o ../results/bowtie2/${i}_sorted.bam ../results/bowtie2/${i}.bam
		#echo ${i}
	done

Remove duplicate by samtools markdup

VAR=$(tail -n +2 ../meta_data/SraRunTable.txt | cut -d ',' -f 1)

for i in ${VAR}
	do
		
		samtools markdup -r ../results/bowtie2/${i}_sorted.bam ../results/bowtie2/${i}_unique.bam
		#echo ${i}
	done

Filter reads with low mapping quality

VAR=$(tail -n +2 ../meta_data/SraRunTable.txt | cut -d ',' -f 1)

for i in ${VAR}
	do
		
		samtools view -q 40 ../results/bowtie2/${i}_unique.bam -o ../results/bowtie2/${i}_mapq40.bam
		#echo ${i}
	done

Merge bamfile

samtools merge -r Oct4_dox.bam SRR3997985_mapq40.bam SRR3997986_mapq40.bam SRR3997987_mapq40.bam
samtools merge -r Smad3_dox.bam SRR3997988_mapq40.bam SRR3997989_mapq40.bam SRR3997990_mapq40.bam
samtools merge -r Smad3.bam SRR3997993_mapq40.bam SRR3997994_mapq40.bam SRR3997995_mapq40.bam
samtools merge -r InputDox.bam SRR3997991_mapq40.bam SRR3997992_mapq40.bam

Remove blacklisted region

Peak calling

macs2 callpeak -t ../results/bowtie2/SRR3997985_unique.bam -c ../results/bowtie2/InputDox.bam -f BAM -g mm –outdir ../macs2 -n Oct4_dox_R1 -q 0.001 macs2 callpeak -t ../results/bowtie2/SRR3997986_unique.bam -c ../results/bowtie2/InputDox.bam -f BAM -g mm –outdir ../macs2 -n Oct4_dox_R2 -q 0.001 macs2 callpeak -t ../results/bowtie2/SRR3997987_unique.bam -c ../results/bowtie2/InputDox.bam -f BAM -g mm –outdir ../macs2 -n Oct4_dox_R3 -q 0.001 macs2 callpeak -t ../results/bowtie2/SRR3997988_unique.bam -c ../results/bowtie2/InputDox.bam -f BAM -g mm –outdir ../macs2 -n Smad3_dox_R1 -q 0.001 macs2 callpeak -t ../results/bowtie2/SRR3997989_unique.bam -c ../results/bowtie2/InputDox.bam -f BAM -g mm –outdir ../macs2 -n Smad3_dox_R2 -q 0.001 macs2 callpeak -t ../results/bowtie2/SRR3997990_unique.bam -c ../results/bowtie2/InputDox.bam -f BAM -g mm –outdir ../macs2 -n Smad3_dox_R3 -q 0.001 macs2 callpeak -t ../results/bowtie2/SRR3997991_unique.bam -c ../results/bowtie2/Input.bam -f BAM -g mm –outdir ../macs2 -n Smad3_R1 –broad -q 0.001 macs2 callpeak -t ../results/bowtie2/SRR3997992_unique.bam -c ../results/bowtie2/Input.bam -f BAM -g mm –outdir ../macs2 -n Smad3_R2 –broad -q 0.001 macs2 callpeak -t ../results/bowtie2/SRR3997993_unique.bam -c ../results/bowtie2/Input.bam -f BAM -g mm –outdir ../macs2 -n Smad3_R3 –broad -q 0.001

Bamcoverage

bamCompare --operation reciprocal_ratio --effectiveGenomeSize 2150570000 -b1 Oct4_dox.bam -b2 InputDox.bam -o Oct4Dox.bw
bamCompare --operation reciprocal_ratio --effectiveGenomeSize 2150570000 -b1 Smad3_dox.bam -b2 InputDox.bam -o Oct4Dox.bw

computeMatrix

computeMatrix reference-point --referencePoint center --sortRegions descend -a 2000 -b 2000 -R Oct4_dox_R1_summits.bed -S ../results/bowtie2/Oct4Dox.bw -o Oct4Dox.mat

plotHeatmap