Truseq stranded mrna library preparation

Pharyngeal mesoderm/ hearts of E8.75 (6 somites) and OFT+RV of E10.5 control and Isl1 KO or hypomorphic embryos were dissected and stored at −80 °C in Qiazol. Total RNA from single embryos was extracted using Rneasy Microarray Tissue Kit (Qiagen #73304). Control, Baf60c KD and Isl1 KO Nkx2.5-GFP ES cell lines were differentiated into cardiac progenitors. Nkx2.5-GFP positive cells from two different control and Isl1^−/− clones or control and Baf60 KD ESCs pools were sorted and RNA was isolated using RNeasy microarray kit (Qiagen #73304). The integrity of RNA was assessed on Bioanalyzer 2100 (Agilent). For RNA-Seq of control, Isl1 hypomorphic and Isl1^−/− tissue samples, 100 ng of total RNAs was used for library preparation and sequenced on BGISEQ-500 or on NextSeq500 (Illumina), respectively. For RNA-Seq of control, Isl1^−/− and Baf60c KD CPCs, 1 µg RNA was used as input for Truseq Stranded mRNA library preparation (Illumina). Sequencing was performed on NextSeq500 (Illumina) using V2 chemistry.

RNA was extracted from snap-frozen colonic mouse tissue (Qiagen RNeasy mini kit cat # 74106, Venlo, The Netherlands) and RNA integrity and quantity was checked with Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). RNA sequencing and preparative techniques were performed by the Genomics Core (UZ Leuven, http://gc.uzleuven.be/). TruSeq stranded mRNA library preparation (Illumina, San Diego, CA, USA) was performed according to the manufacturer's guidelines. Sequencing was performed on the HiSeq2500 platform with a sequencing depth ranging from 9 to 21 M reads per sample (Illumina). The adaptors were trimmed from the reads with the use of ea_utils v1.1.2. Reads that were shorter than 25 bp after adaptor trimming were removed. The pre-processed reads were aligned to the reference genome of Mus musculus (Mm10) with TopHat v2.0.13. Counting of the reads was performed with HTSeq46 (link) or CuffQuant47 (link). Pathway analysis was performed with Ingenuity Pathway Analysis (IPA, Qiagen). Raw data have been deposited in BioProject with the accession code PRJNA374413 (https://www.ncbi.nlm.nih.gov/bioproject/374413).

Total RNA was isolated from human cardiac fibroblasts seeded on tissue culture plastic, intact ECM scaffolds, and neutralized ECM scaffolds using RNeasy Mini Kit (Qiagen, Germany), per manufacturer’s instructions. RNA quality was assessed with the Agilent 2200 Tapestation RNA (RIN) assay (Agilent, CA, USA). 30 µL of total RNA per sample were used for cDNA library preparation [TruSeq Stranded mRNA Library Preparation (Illumina, CA, USA). RNA sequencing data (535 M reads) was generated using a 75 cycle high-output kit on an Illumina NextSeq500 (Illumina, CA, USA). RNA sequencing reads pseudoaligned to the human NCBI RefSeq transcript database dated January 2017, using Kallisto 0.42.4^{37 (link),38} . Sleuth was used for differential gene expression using a linear model containing two terms: the nominal scaffold factor and the patient from which each sample was derived^{39 (link)}. Genes passing the Likelihood Ratio Test with Benjamin-Hochberg (false discovery rate) corrected p-values and Wald test (<0.05) were considered differentially expressed. Differentially expressed genes were annotated and analyzed for enrichment using Ingenuity Pathway Analysis (Qiagen, CA, USA).

RNA was extracted from HCT116 Ki-67-AID in presence or absence of Ki-67 and extracted using the Monarch Total RNA Miniprep Kit (New England Biolabs, Hitchin, UK) according to manufacturer’s protocol. RNA samples were sent to Macrogen (Japan). Macrogen Europe BV constructed libraries using Illumina TruSeq stranded mRNA library preparation with Ribozero rRNA depletion. Sequencing was performed with a Novaseq 6000 platform, at 50 M paired-end reads per sample.
The paired-end raw reads were mapped to the human reference genome GRCh38 using the annotations from GENCODE 28 [72 (link)] with HISAT2 under standard conditions.
The resulting alignments were filtered for high-quality hits using SAMtools v0.1.19 [73 (link)] with a minimum selection threshold score of 30. HTSEQ was used to assemble the mapped reads into transcripts and quantify their expression levels.
Deseq2 was used to identify differentially transcribed genes between samples. The differential expression was expressed in the form of log2 fold change between the sample and control and deemed statistically significant by a lower p value of 0.05.
Functional enrichment was analysed using String (string-db.org), while Venn diagrams were performed in the open software FunRich. Volcano plots were performed using the ggplot package in R v3.5.0.