For Fig. 4e–j and Figure S2c–e , population RNA-seq data were generated as previously described1 (link). Briefly, total RNA isolation was performed using ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen) according to the manufacturer’s instructions. Total RNA was used for quality controls and for normalization of starting material. cDNA libraries were generated using 1 ng of total RNA for Neo1-deficient/Wt HSCs using the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech) according to the manufacturer’s indications. Sequencing was performed using the HiSeq2000 device (Illumina).
Hiseq2000 device
Manufactured by Illumina
Sourced in United States
The HiSeq2000 is a high-throughput DNA sequencing device manufactured by Illumina. It is designed to perform massively parallel sequencing of DNA samples, generating large volumes of sequencing data efficiently.
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Lab products found in correlation
Smarter ultra low rna kit for sequencing, by Illumina (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Miseq device, by Illumina (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
Truseq rna library prep kit, by Illumina (1 mentions)
Nextera kit, by Illumina (1 mentions)
Rnase free dnase set, by Qiagen (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
Ingenuity pathway analysis software, by Qiagen (1 mentions)
Biorobot ez1, by Qiagen (1 mentions)
Ez1 rna universal tissue kit, by Qiagen (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Nebnext ultra rna library prep kit, by New England Biolabs (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Ab5821, by Abcam (1 mentions)
Ab4729, by Abcam (1 mentions)
10 protocols using hiseq2000 device
1
RNA-seq Protocol for Neo1-deficient HSCs
2
Whole Genome Sequencing of Mycobacterium tuberculosis
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WGS was performed as detailed elsewhere (Perez-Lago et al., 2014 (link)) using a HiSeq 2000 device and a Miseq device (Illumina), which generated 101-51–bp paired-end reads. We mapped the reads for each strain using the Burrows-Wheeler Aligner and the ancestral MTB genome as detailed elsewhere (Comas et al., 2013 (link)). SNP calls were made with SAMtools and VarScan (coverage of at least 10×, mean SNP mapping quality of 20). The genome was compared between strains using an in-house script written in R.
3
RNA-seq Analysis of Cyanidioschyzon merolae
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Cyanidioschyzon merolae cells were grown until late log-phase. RNA was isolated using RNeasy Plant Mini Kit (Qiagen), following a standard protocol. cDNA synthesis and DNase treatment were performed using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) with oligo(dT) primers, according to a standard manual. The quality and concentration of samples were measured spectrometrically using Nanodrop 1000 (Thermo Scientific) and electrophoretically using Bioanalyzer 2100 (Agilent Technologies). Libraries were constructed using TruSeq RNA Library Prep kit (Illumina) with gel-free library purification based on Agencourt AMPure XP beads (Beckman Coulter, Inc.). The samples were sequenced on HiSeq 2000 device (Illumina Inc.). Paired-end raw sequencing reads were analyzed using Galaxy implementations (usegalaxy.org) of FastQC program and Tuxedo protocol (Trapnell et al., 2012 (link)). The new annotation from C. merolae Genome Project v3 was employed as a reference. The correlation between replicates in RNA-seq experiment was scored using Pearson correlation and shown in Supplementary Figure S4 . The data was deposited in the Gene Expression Omnibus, under the series GSE93912.
4
Assembly and Analysis of Wheat Chromosome 4AL
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A CSS assembly of CS chromosome arms 4AS (4AS‐CS) and 4AL (4AL‐CS) were acquired from Internation Wheat Genome Sequencing Consortium (IWGSC, 2014 ). Two sequencing libraries of DNA amplified from the 4AL‐TM telosome were constructed using a Nextera kit (Illumina, San Diego, CA) with the insert size adjusted to 500 and 1000 bp. The resulting clones were sequenced as paired‐end reads by IGA (Udine, Italy) using a HiSeq 2000 device (Illumina). The 4AL‐TM reads were assembled with SOAPdenovo2 software, applying a range of k‐mers (54–99, with a step size of 3) to select the assembly with the highest coverage and the largest N50. Assembled scaffolds (k‐mer of 69, minimum length 200 bp) were chosen for further analysis (Table 1 ).
5
Total RNA Isolation and Sequencing
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Total RNA isolation was performed from the indicated populations using ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen) according to the manufacturer’s instructions. DNase treatment was performed using RNase-free DNase Set (QIAGEN). Total RNA was used for quality controls and for normalization of starting material (Figure S2 ). cDNA-libraries were generated with 10 ng of total RNA using the SMARTer Ultra Low RNA Kit for Illumina Sequencing (Clontech) according to the manufacturer’s indications. Of note, 12 cycles were used for the amplification of cDNA, respectively. Paired-end adaptors were applied to each population. Sequencing was performed with the HiSeq2000 device (Illumina) and one sample per lane. Quality controls before and after sequencing and schematic overview of sampling workflow are shown in Figures S1 and S2 .
6
Microarray and RNA-seq Analysis of AML Subpopulations
7
Transcriptome Sequencing and Differential Expression Analysis
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Transcriptome sequencing libraries were prepared from isolated total RNA using the TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA), and single-read 50 bp sequencing was performed in a HiSeq-2000 device (Illumina). Reads were then trimmed by removing stretches of bases with a quality score <30 at their ends, and subsequently mapped using Tophat2.0.6 against the hg19 assembly of the human genome.17 (link) Finally, differential expression was quantified using DESeq2 and Cuffdiff 2.0, and subjected to diverse testing corrections.18 (link),19 (link) Genes with a q-value <0.05 were considered differentially expressed. Principal component analysis plots and heat maps were created in R using the FactoMineR and pheatmap packages, respectively. For gene set enrichment analysis (GSEA, Broad Institute, Boston, MA, USA),20 (link) the fragments per kilobase of transcript per million mapped reads (FPKM) values obtained from Cuffdiff 2.0 for the different samples were compared either with the gene sets contained in the Molecular Signature Database or with self-made gene lists. The Signal2Noise metric and 1000 gene set-based permutations were applied to all analyses. Ingenuity pathway analysis software (Qiagen, Hilden, Germany) was used to predict the potential extrinsic factors capable of generating the different RNA sequencing data sets.
8
RNA Extraction and Sequencing of Naegleria fowleri
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RNA was extracted from 107
N. fowleri trophozoites cultivated in Nelson’s medium using the EZ1 RNA Universal Tissue Kit (Qiagen) and the EZ1 BioRobot (Qiagen). Trophozoites were resuspended in 750 μl of QIAzol lysis reagent (Qiagen), followed by disruption and homogenization by operating a TissueLyser at 25 Hz for 3 min. After incubation for 5 min at room temperature, 150 μl chloroform (Grogg, Stettlen, Switzerland) was added to the homogenized samples. The mixture was then centrifuged for 15 min at 12,000 g at 4°C, and the upper aqueous phase was used as the starting material for RNA isolation with the EZ1 BioRobot, according to manufacturer’s protocol. Quantification and examination of the total RNA integrity was performed with the Agilent 2100 Bioanalyzer system. Four micrograms of high-quality RNA was sent to the Next Generation Sequencing Platform of the University of Bern for paired-end sequencing by the Illumina HiSeq 2000 device.
The reads from RNA sequencing have been deposited in DDBJ/EMBL/GenBank under accession SRX553040.
N. fowleri trophozoites cultivated in Nelson’s medium using the EZ1 RNA Universal Tissue Kit (Qiagen) and the EZ1 BioRobot (Qiagen). Trophozoites were resuspended in 750 μl of QIAzol lysis reagent (Qiagen), followed by disruption and homogenization by operating a TissueLyser at 25 Hz for 3 min. After incubation for 5 min at room temperature, 150 μl chloroform (Grogg, Stettlen, Switzerland) was added to the homogenized samples. The mixture was then centrifuged for 15 min at 12,000 g at 4°C, and the upper aqueous phase was used as the starting material for RNA isolation with the EZ1 BioRobot, according to manufacturer’s protocol. Quantification and examination of the total RNA integrity was performed with the Agilent 2100 Bioanalyzer system. Four micrograms of high-quality RNA was sent to the Next Generation Sequencing Platform of the University of Bern for paired-end sequencing by the Illumina HiSeq 2000 device.
The reads from RNA sequencing have been deposited in DDBJ/EMBL/GenBank under accession SRX553040.
9
Transcriptome Analysis of Wheat Ploidy Series
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The youngest fully expanded leaf at development stage 5 [44 (link)] was collected from three-five individuals of the parental, allotriploid and allohexaploid plants, snap-frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted from the leaf tissue using an RNAprep Pure Plant kit (TIANGEN, Beijing, China), according to the manufacturer’s protocol. The integrity of the extracted RNA was validated using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The RNA was used to construct and sequence 13 RNA-Seq libraries: two each from the AS2255 × AS60 and LDN × AS60 allotriploids, the AS2255 × AS60 and LDN × AS60 allohexaploids, AS2255 and AS60, and one from LDN. Paired end sequencing libraries (average insert size: 200 bp) prepared using a NEBNext® Ultra™ RNA Library Prep kit (New England Biolabs) and sequenced using a HiSeq2000 device (Illumina, San Diego, CA, USA) according to the manufacturer’s standard protocols. Raw RNA reads were de-multiplexed using bcl2fastq v1.8.4 (support.illumina.com/downloads/bcl2fastq_conversion_software_184.html). All contaminants and low quality reads were removed by enforcing a Q30 threshold of 80% and a maximum of 0.2% ambiguous base calls.
10
ChIP-seq Protocol for Fibrillarin and H3K27ac
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ChIP sequencing was performed as previously described [39 (link)] starting from 10*107 REH cells. Nuclei were isolated and chromatin was purified by chemical lysis and the purified chromatin was fragmented to 200-300 bp fragments by sonication (Covaris, S220, Focused-ultrasonicator). Chromatin immunoprecipitation was performed by incubation of the chromatin fraction overnight with 100 μl of protein-A coated beads (Thermo-Scientific) and 10 μg of fibrillarin-specific (Abcam, ab5821) or H3K27ac-specific antibodies (Abcam, ab4729). The next day, beads were washed to remove non-specific binding events and enriched chromatin fragments were eluted from the beads, followed by reverse cross-linking by incubation at 65°C overnight. DNA was subsequently purified by phenol/chloroform extraction and used for library preparation and sequencing using an Illumina Hiseq 2000 device (San Diego, CA, USA). Raw sequencing data were mapped to the human reference genome (GRCh37/h19) using Bowtie. Peak calling was performed using MACS 1.4. The ChIP-seq data were deposited in the GEO database (GSE79873).
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