CELR-D-22-00015

• Repository that contains the analysis from Li et al. Cell Regeneration, 2022.
• All ChIP-seq (GSE203304) and RNA-seq (GSE203305) data in this paper are accessible at GEO Accession viewer under the number: GSE203306.

I. Upstream analysis

• RNA-seq_analysis : RNA-seq_analysis.sh
  RNA-seq data is applied quality and adapter trimming by Trim Galore v0.6.4_dev and mapped to the mm9 reference genome using STAR v2.7.2d. And we performed reads summarization for genomic features via featureCounts v2.0.0.

• ChIP-seq_analysis : ChIP-seq_analysis.sh
  Adapters and low-quality bases were removed using Trim Galore v0.6.4_dev. ChIP-seq reads were mapped to the mm9 reference genome using Bowtie2 v2.3.5.1. Samtools v1.10 was used to filter and convert file formats. To remove PCR duplicate reads, which might occur false positive results, MarkDuplicates (v4.1.4.1, Picard) was used to locate and tag duplicate reads. And sequencing duplicates were removed using the --REMOVE_SEQUENCING_DUPLICATES options. ChIP-seq peaks were called using MACS2 v2.2.6.

• Repetitive Elements binding analysis
  For the repetitive Elements binding analysis, the reads were then aligned to the mm9 genome assembly using STAR v2.7.2d with the options --alignIntronMax 1 and --alignEndsType EndToEnd as previously reported. The parameter --outFilterMultimapNmax 1 was applied to include only the uniquely mapped reads. Duplicate reads were then removed using MarkDuplicates (v4.1.4.1, Picard). Replicate samples were merged using the Samtools v1.10.

II. Downstream analysis

• The raw read counts normalization and differential expression were determined by RNA-seq_rif1.R.

  coding_genes.R is for further analysis of genes
  repeats.R is for further analysis of repeats

• Correlation among different samples is analyzed via CalculateCorrelation.R and plotCorrHeatmap.R is used to plot correlation heatmap.

• Principal Component Analysis (PCA) is calculated the foldchange (vs. control/wild type) between the samples with the depletion of the indicated genes to control (or wild type) samples via PCAanalysis.R.

• GOanalysis.R is used to perform GO analysis.

• Single locus repeats RNA-seq analysis needs single locus position annotation file to annotate the reads signal in genome.

  featureCounts sample.repeats.Aligned.sortedByCoord.out.bam \
                -a ~/project/rif1/mm9_repeats_single_locus.gtf \
                -g gene_id2 -o sample.counts -T 40
  

III. Quality Control

All data in this article has been quality controlled, the qc reports are as follows:

• ChIP-seq_multiqc_report
• RNA-seq_multiqc_report