Computational protocol: Constitutively Active SMAD2/3 Are Broad Scope Potentiators of Transcription Factor Mediated Cellular Reprogramming

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Protocol publication

[…] Reads were quality-checked using FastQC and trimmed using Trimmomatic to remove adapters and low quality bases (). Reads were aligned to the mm10 assembly of the mouse genome using Bowtie 2 with the very-sensitive option (). Duplicate reads were removed using Picard Tool’s MarkDuplicates command and were filtered for MAPQ > = 40 using SAMtools (). Reads aligned to ENCODE blacklist regions were removed using BEDTools (). Peaks were called using MACS 2 on both individual and merged sample replicates against merged control replicates (). For narrow peak factors, such as OCT4, peaks were called with the -q 0.01 and —call-summits specified. For broad peak factors, such as SMAD3, peaks were called with the–broad and–broad-cutoff 0.1 options specified. Differentially-bound sites were identified using the DiffBind software package from the Bioconductor project (). A FDR ≤ 0.1 and absolute FC > = 1 were used as the significance threshold. Merged control replicates were used for contrast against each individual sample replicate. The deepTools suite was used to generate normalized input-subtracted read coverage and heatmap scores using the bamCompare and plotHeatmap commands, respectively. Normalized input-subtracted read coverage was generated from merged sample and control replicates with the–ratio subtract and–normalizeTo1x 2150570000 option specified. Heatmaps were generated using the normalized input-subtracted read coverage and peaks called from the merged sample replicates. [...] Reads were quality-checked using FastQC and trimmed using Trimmomatic to remove adapters and low quality bases (). Reads were aligned to the mm10 assembly of the mouse genome using Bowtie 2 () with the–very-sensitive and -X 2000 options specified. Duplicate reads were removed using Picard Tool’s MarkDuplicates command and were filtered using SAMtools for MAPQ > = 30 and the properly-paired (0x2) flag (). Reads aligned to the ENCODE blacklist and mitochondrial blacklist regions were also removed using BEDTools (). The deepTools suite was used to generate normalized read coverage and heatmap scores using the bamCoverage and plotHeatmap commands, respectively (). Normalized read coverage was generated from merged sample replicates with the–normalizeTo1x 2150570000 option specified. Heatmaps were generated using the normalized read coverage and peaks called from the merged sample replicates. The data of one of the ATAC-seq replicates had low read counts and demonstrated abnormal features as assessed by PCA and hierarchical clustering, clustering independent of all other samples including the starting MEF population and resulting iPSC cells, and was therefore removed from the analysis. […]

Pipeline specifications

Software tools FastQC, Trimmomatic, Bowtie, Picard, SAMtools, BEDTools, deepTools
Application ATAC-seq analysis
Organisms Homo sapiens