7500 chip

Strand-specific libraries were generated using the TruSeq RNA access sample preparation kit according to the manufacturer's instructions (Illumina, Part # 15049525 Rev. B). The FF RNA was fragmented, random primed and reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen, part # 18064-014) with the addition of Actinomycin D. Second strand synthesis was performed using Polymerase I and RNaseH with replacement of dTTP for dUTP. The generated cDNA fragments were 3' end adenylated and ligated to Illumina sequencing adapters and subsequently amplified by 15 cycles of PCR. The libraries were analyzed on a 2100 BioAnalyzer using a 7500 chip (Agilent, Santa Clara, CA). Capture pools of 200 ng per sample were compiled based on library yield and unique adapter sequence, where after target enrichment of the exome is performed. The captured fragments were amplified by a 2nd amplification of 10 PCR cycles. The captured fragments were assessed on a 2100 BioAnalyzer, diluted and equimolar pooled into a 10 nM multiplex sequencing pool, containing 12 samples per pool.

Fresh-frozen tumor samples were sectioned, collected for RNA preparation and in part subjected to tumor percentage evaluation by revision of HE stained coupes by senior head and neck pathologist Dr. S.M. Willems. Only samples with a tumor percentage of >40% proceeded to RNA-sequencing. RNA was isolated using the AllPrep DNA/RNA mini kit (Qiagen). Quality and quantity of total RNA was assessed by the 2100 Bioanalyzer using a Nano chip (Agilent, Santa Clara, CA). Only total RNA samples having RIN>7 were used for library preparation. Strand-specific libraries were generated using the TruSeq Stranded mRNA sample preparation kit (Illumina Inc., San Diego, RS-122-2101/2) according to the manufacturer's instructions (Illumina, Part # 15031047 Rev. E). The libraries were analyzed on a 2100 Bioanalyzer using a 7500 chip (Agilent, Santa Clara, CA), diluted and pooled equimolar into a 10 nM multiplex sequencing pool and sequenced with 65 base single reads on a HiSeq2500 using V4 chemistry (Illumina Inc., San Diego). Reads were mapped against the GRCh38 human genome using TopHat2.1 (70 (link)), with options “fr-firststrand,” “transcriptome-index,” and “prefilter multi-hits.” Read counts were determined using HTSeq-count (71 (link)) with options “stranded” and mode “union.”

PBMCs from 3 healthy donors were stained with the appropriate antibodies (Supplementary Fig. 1a) and within the live CD3⁻CD19⁻HLA-DR⁺ population, pDC (CD11c⁻CD14⁻CD303⁺), cDC1 (CD11c⁺CD14⁻CD141⁺), cDC2 (CD11c⁺CD14⁻CD1c⁺), and moDC (CD11c⁺CD14⁺CD1c⁺CD206⁺) were sorted. Then cells were washed in ice-cold PBS and resuspend in buffer RLT (Qiagen). Total RNA isolation was performed according to manufacturer’s protocol using the RNeasy MinElute Cleanup Kit (Qiagen). Quality and quantity of the total RNA were assessed on a 2100 Bioanalyzer using a Nano chip (Agilent). Only RNA samples having an RNA Integrity Number (RIN) > 8 were subjected to library generation. Strand-specific cDNA libraries were generated using the TruSeq Stranded mRNA sample preparation kit (Illumina) according to the manufacturer’s protocol. The libraries were analyzed for size and quantity of complementary DNAs (cDNAs) on a 2100 Bioanalyzer using a 7500 chip (Agilent), diluted and pooled in equimolar ratios into a multiplex sequencing pool. The libraries were sequenced as 65 base single reads on a HiSeq2500 with V4 chemistry (Illumina).

Sections (30 µm thick) were cut from the frozen tumor tissues for RNA isolation. Total RNA was extracted using the mirVana miRNA isolation kit (Ambion, USA) according to the manufacturer’s protocol until the end of F1. Quality and quantity of the total RNA was assessed by the 2100 Bioanalyzer (Agilent, USA). Total RNA samples having RIN >8 were subjected to library generation.
Strand-specific libraries were generated using the TruSeq Stranded mRNA sample preparation kit (Illumina, USA; RS-122-2101/2) according to the manufacturer’s instructions (Illumina, Part # 15031047 Rev. E). 3′ adenylated and adapter ligated cDNA fragments were subject to 12 cycles of PCR. The libraries were analyzed on a 2100 Bioanalyzer using a 7500 chip (Agilent, Santa Clara, CA), diluted and pooled equimolarly into a multiplexed, 10 nM sequencing pools and stored at −20 °C. Strand-specific cDNA libraries were sequenced with 100 base paired-end reads on a HiSeq2500 using V4 chemistry (Illumina).

Polyadenylated RNA was purified by oligo-dT beads for both nuclear and cytoplasmic fractions, reverse transcribed (SuperScript II Reverse Transcriptase, Invitrogen, # 18064–014) and constructed into strand-specific libraries using the TruSeq Stranded mRNA sample preparation kit (Illumina Inc., San Diego, RS-122-2101/2) according to the manufacturer's instructions (Illumina, # 15031047 Rev. E). The generated cDNA fragments were 3' end adenylated and ligated to Illumina Paired-end sequencing adapters and subsequently amplified by 12 cycles of PCR. The libraries were analyzed on a 2100 Bioanalyzer using a 7500 chip (Agilent, Santa Clara, CA), diluted and pooled equimolar into a 12-plex for each replicate and subjected to sequencing with 50 base single reads on a HiSeq2500 using V4 chemistry (Illumina Inc., San Diego). The two replicates were sequenced in two separate lanes. The total reads for the two replicates are 182,747,266 and 177,771,796, with even reads distribution for each time point.
Reads were mapped first to the transcriptome of S. cerevisiae (Saccharomyces_cerevisiae.R64-1-1.78) and then to the transcriptome of D. melanogaster (Drosophila_melanogaster.BDGP5.77) by Tophat [76 (link)].

Genomic DNA of MCF10A and MCF10A-YAP^5SA cells transduced with sgRNAs was isolated and the concentration was measured. Five hundred nanograms of the genomic DNA was used for PCR-amplification of the enhancer region. We performed a two-step PCR by introducing the P5 adapter sequence in the first PCR and P7 adapters with the indexes in the second PCR. After the second PCR, the libraries were purified with CleanNA beads (GC Biotech) and quantified on the 2100 Bioanalyzer using a 7500 chip (Agilent). Equimolar amounts of each sample were taken for samples ran on the same lane. Deep sequencing was performed with single reads of 150 bp on the Mi-Seq system with Mi-Seq reagent v2 Nano kit. Sequenced reads were aligned to the amplified enhancer region using Bowtie. Bam files were analyzed to count the number of mutations (mismatches, insertions, or deletions) identified at each location in the enhancer regions.