Dna analysis screentape

RNA was extracted from paired specimens as described above. mRNA quality was assessed for library preparation using DNA Analysis ScreenTape (Agilent Technologies). Libraries for cDNA were generated using either TrueSeq (for frozen samples) or TrueSeq RNA Access (for FFPE-preserved samples) library kits (Illumina). Libraries were sequenced using either the Illumina HiSeq4000 platform, or NovaSEQ 6000 platform, both with paired-end reads (2 × 150). Forty million reads/sample were collected on average. Resulting sequences were filtered and trimmed, removing low-quality bases (Phred score <15), and analyzed using a custom computational pipeline consisting of open-source trimmomatic (RRID:SCR_011848), gSNAP (RRID:SCR_005483), and Cufflinks (RRID:SCR_014597). R was used for differential gene expression alignment and discovery. Data were then analyzed using either GSEA 3.0 (The Broad Institute, RRID:SCR_005724) or Ingenuity Pathway Analysis (IPA; Qiagen, RRID:SCR_008653).

E10.5 FNP and MxP bulk tissue from three control and three Tfap2^Δ/NCKO littermates were processed for RNA-seq as previously described (Van Otterloo et al., 2016 (link)). After PCR-genotyping, samples stored in RNAlater (ThermoFisher) were processed for RNA (Norgen Biotek) and further purified with the RNAeasy kit (Qiagen). mRNA quality was determined with DNA Analysis ScreenTape (Agilent Technologies), and cDNA libraries were generated using the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina). Samples were sequenced on the Illumina HiSeq2500 platform as 150-bp single-end reads. Library construction and sequencing was carried out by the Genomics and Microarray Core on the University of Colorado Anschutz Medical Campus.