Hi All! I need a bit of help getting my head around this problem.
We did TERF2 gene silencing (3 biological replicates, both control case ie 3 sets) in HT1080 fibrosarcoma cell line using shRNA that binds with full complementarity to its 7th exon. In WB we see 30-40% protein level downregulation (in two sets) and 20-30% in (3rd set). qPCR also shows 60% silencing in 2 sets and 40% silencing in one set.
However, when I performed Differential Expression analysis, I couldn't see TERF2 downregulated significantly. My log2FC was -0.34249 with a p-value of 0.075 and p-adj 0.721. When I compared raw read counts the difference between control &case in all three replicates was about 200-300 reads. What could have gone wrong? Out data is paired end RNAseq
We suspected that there might be a novel transcript that has not been reported so far and isn't degraded and might be contributing to increased read numbers. For that, we checked the Isoform percentage values that RSEM produces. We found that majority of the transcripts were coming from. I think TERF2-201 is going down while TERF-2-206 is coming up, which is visible in two sets, but I am not sure.
In addition to the questions above, I would like to know if the majority of reads (~70%) are coming from a single transcript, is it true that if we count reads exon-wise (within exon boundaries), the number of reads (or distribution) will be similar across all exons.
rsem-prepare-reference --gtf /path to/gencode.v33.annotation.gtf --star --star-path ~/anaconda3/bin/ /path to/GRCh38.primary_assembly.genome.fa /home/parashar/scratch/name_of_output_file 2> rsem.stderr rsem-calculate-expression --star-output-genome-bam --strandedness reverse -p 32 --calc-pme --calc-ci --keep-intermediate-files --append-names --sort-bam-by-coordinate --paired-end --estimate-rspd --star --star-path ~/anaconda3/bin/ ~/trimmomatic/paired/Contr1_S15_L004_R1_p.fastq ~/trimmomatic/paired/Contr1_S15_L004_R2_p.fastq ~/rnaseq/rsem ~/Contr1_S15_L004 2> ~/Contr1_S15_L004.stderr
The DESeq codes are here Please Note the DE-Seq didn't account for batch correction.I am on it. The HTSeq-count was used for raw feature estimation in union mode. The RSEM was used in STAR mode. The Star genomic alignment was used for downstream analysis Alignment Stats from STAR Aligner