The cells were assigned to one of the three groups: control group, COL28 negative control group (COL28-NC group), and COL28 overexpression group (COL28-OE group). The extraction, library building, and sequencing of total RNA of cells were performed by Aimer Gene (Xiamen) Biotechnology Co., Ltd. TRIzol (Invitrogen) was employed to extract the total RNA from the samples, NanoDrop 2000 (Thermo Fisher Scientific) to evaluate the quality and concentration of the RNA, and QIAxcel advanced system (Qiagen, advanced) to assess RNA integrity. With the help of NEB NextUltra™ RNA Library Prep Kit for Illumina (New England BioLabs, Inc., Ipswich, MA, USA), RNA-seq libraries were created under the recommendations of the manufacturer. Using the Illumina HiSeq 4000 instrument (Illumina, Inc., San Diego, CA, USA), the quality-checked libraries were sequenced, and paired-end reads were produced. Initial processing of the raw data in FASTQ format was done with an internal Perl script. By removing low-quality reads, ploy-N, and reads containing adapters, the clean reads were achieved. For the purpose of assessing the sequencing quality, the GC content and Q20 were determined. Utilizing star (for genome comparison), cufflinks (for expression analysis), and deseq2 (for differential expression), the obtained reads were analyzed [21 (link)].
Hiseq 4000 instrument
Manufactured by Illumina
Sourced in United States, United Kingdom
The HiSeq 4000 instrument is a high-throughput DNA sequencing system designed and manufactured by Illumina. It is capable of generating large volumes of sequencing data quickly and efficiently. The core function of the HiSeq 4000 is to perform massively parallel DNA sequencing using Illumina's proprietary sequencing-by-synthesis technology.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Qiaamp dna mini kit, by Qiagen (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Ribo zero rrna removal kit, by Illumina (3 mentions)
Rneasy micro kit, by Qiagen (3 mentions)
Tapestation 2200, by Agilent Technologies (3 mentions)
Nextera rapid capture enrichment kit cex version coding exome oligos, by Illumina (3 mentions)
Truseq stranded mrna library prep kit, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Truseq rna library prep kit, by Illumina (2 mentions)
Hiseq 3000 4000 sbs kit, by Illumina (2 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (2 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (2 mentions)
Tri reagent, by Merck Group (2 mentions)
C1000 touch pcr instrument, by Bio-Rad (2 mentions)
Rneasy, by Qiagen (2 mentions)
74 protocols using hiseq 4000 instrument
1
RNA-seq Protocol for Differential Expression
2
Stranded RNA Sequencing of Canine Tumors
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Stranded RNA sequencing libraries with insert size 100–300 bp were generated for 33 CTVTs (1208Ta-Dog, 1210Ta-Dog, 1247Ta-Dog, 126Ta-Dog, 131Ta-Dog, 1532Ta-Dog, 24Ta-Dog, 335Ta-Dog, 341Ta-Dog, 355Ta-Dog, 365Tb-Dog, 366Ta-Dog, 410Ta-Dog, 423Ta-Dog, 439Tb-Dog, 459Ta-Dog, 464Ta-Dog, 468Ta-Dog, 550Ta-Dog, 556Tb-Dog, 559Ta-Dog, 560Ta-Dog, 608Ta-Dog, 609Ta-Dog, 645Ta-Dog, 652Ta-Dog, 666Ta-Dog, 683Ta-Dog, 773T1a-Dog, 79Ta-Dog, 809Ta-Dog, 851Ta-Dog and 855Ta-Dog) with the Ribo-Zero ribosomal RNA removal kit (Illumina, San Diego, CA, USA) using standard methods according to the manufacturer’s instructions. The libraries were sequenced with 75-bp paired-end reads on an Illumina HiSeq4000 instrument (Illumina, San Diego, CA, USA) to an average depth of ~168×13 (link). Samples were processed in three batches (Supplementary Data 6A ).
3
Circular RNA Profiling in Hippocampus
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Total RNA was extracted from six hippocampal tissues from each group. The RNA concentration and purity in each sample were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA). RNA integrity was assessed using an Agilent 2100 instrument, with RIN > 7.0. Approximately 5 μg of total RNA was used to deplete ribosomal RNA according to the instructions of the Ribo-Zero™ rRNA Removal Kit (Illumina, San Diego, USA), followed by cDNA library construction. Next, deep sequencing was performed with an Illumina HiSeq 4000 instrument (LC Bio, China) according to the vendor's recommended protocol. First, Cutadapt was used to remove the reads that contained adaptor contamination, low-quality bases and undetermined bases. Then, sequence quality was verified using FastQC. We used Bowtie2 and Hisat2 to map reads to the genome of the species. The remaining reads (unmapped reads) were still mapped to the genome using TopHat fusion. CIRCExplorer2 and CIRI were first used for the de novo assembly of the mapped reads to circular RNAs; then, back splicing reads were identified in unmapped reads using TopHat fusion. All samples generated unique circular RNAs. The differentially expressed circRNAs were selected with log2 (fold change) > 1 or log2 (fold change) < − 1 and with statistical significance (P value < 0.05) using the R package edgeR.
4
DNA Extraction and Sequencing Protocol
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DNA was extracted as previously described [2 (link)]. Genomic DNA for each sample was quantified using the Qubit® 2.0 Fluorometer and was used for library preparation. DNA for intact samples was sheared using a Covaris E220 DNA sonicator to fragments of 500 bp. The DNA libraries for intact samples were made using the TruSeq Nano DNA Library Prep kit (Illumina), whereas the DNA libraries for degraded samples were made using Ovation Ultralow Library System V2 kit (Nugen), according to the manufacturers’ instructions. The amplified libraries were stored at − 20 °C. The pooled libraries were sequenced in an Illumina HiSeq4000 instrument (2 × 150 bp PE reads) (Illumina). A PhiX control library was applied to the sequencing run as a base balanced sequence for the calibration of the instrument so that each base type is captured during the entire run. Samples AF22, AF26 and AF36 were additionally sequenced and scaffolded by PacBio RS II platform (Pacific Biosciences, CA, USA) using a SMRT library. Genomic DNA from the P. vivax samples was extracted from filter paper as previously described [46 (link)].
5
Sequencing and Data Processing Protocol
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DNAseq libraries were prepared from total gDNA using the Celero PCR workflow with an enzymatic fragmentation kit from Tecan (Männedorf, Switzerland). DNAseq libraries were loaded on an Illumina S2 flow cell and sequenced on the Illumina Novaseq 6000 instrument (Illumina, San Diego, CA, USA) as 2 x 151 bp paired-end reads.
Hi-C libraries were prepared from 0.2 g of frozen leaves using the Proximo Hi-C Kit following the manufacturer’s instructions (Phase Genomics, Seattle, WA, USA) and sequenced on an Illumina HiSeq 4000 instrument (Illumina) as 2 x 151 bp paired-end reads.
mRNA stranded libraries were prepared from 500 ng of total RNA using the Tecan Universal Plus mRNA-Seq library preparation kit with NuQuant® and sequenced on an Illumina HiSeq 4000 instrument as 2 x 151 bp paired-end reads.
Illumina raw reads generated from DNAseq libraries and Hi-C libraries were cleaned using fastp 0.23.2 (--length_required 75 --low_complexity_filter) (Chen et al., 2018 (link)).10.1038/s41597-021-00968-x
Hi-C libraries were prepared from 0.2 g of frozen leaves using the Proximo Hi-C Kit following the manufacturer’s instructions (Phase Genomics, Seattle, WA, USA) and sequenced on an Illumina HiSeq 4000 instrument (Illumina) as 2 x 151 bp paired-end reads.
mRNA stranded libraries were prepared from 500 ng of total RNA using the Tecan Universal Plus mRNA-Seq library preparation kit with NuQuant® and sequenced on an Illumina HiSeq 4000 instrument as 2 x 151 bp paired-end reads.
Illumina raw reads generated from DNAseq libraries and Hi-C libraries were cleaned using fastp 0.23.2 (--length_required 75 --low_complexity_filter) (Chen et al., 2018 (link)).10.1038/s41597-021-00968-x
6
RNA-Seq Library Preparation and Sequencing
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The RNA-Seq transcriptome libraries were prepared using a TruSeq RNA sample preparation kit from Illumina (San Diego, CA, USA) with 5 μg total RNA. mRNA was then isolated according to the polyA selection method using oligo(dT) beads and fragmented in fragmentation buffer. Next, double-stranded cDNA was then synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) with random hexamer primers (Illumina). The synthesized cDNA was subsequently subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina's library construction protocol. Libraries were size-selected for cDNA target fragments of 200–300 bp on 2% Low Range Ultra Agarose (Bio-Rad, Hercules, CA, USA) followed by PCR amplification using Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA) for 15 PCR cycles. After quantification using a TBS380 Fluorometer (Turner Biosystems Inc., Sunnyvale, CA, USA), a paired-end RNA-Seq library was sequenced with an Illumina HiSeq 4000 instrument (2 × 150 bp read length). The sequencing data were deposited in the NCBI/SRA database (Bioproject: PRJNA422178; BioSample: SAMN08166800; Sequence Read Archive Database under accession number SRP126885).
7
CUT&Tag Chromatin Profiling Protocol
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CUT&Tag assay was performed as per literature.49 Briefly, 100,000 cells were harvested, washed and mixed with activated concanavalin A‐coated magnetic beads (Bangs Laboratories) at room temperature for 15 min. The mix was resuspended in 100 μl DIG‐wash buffer consisting of 2‐mM EDTA and primary antibody (1:50 dilution) and incubated overnight at 4℃. The secondary antibody was added to the cells at a 1:50 dilution and incubated at room temperature for about 1 h. After washing, 100 μl of pA‐Tn5 adapter complex (∼40 nM) was added into the cells and incubated at room temperature for about 1 h. After washing, the cells were resuspended in Tagmentation buffer (50 μl) and incubated at 37°C for 1 h. Then, proteinase K treatment was used at 55°C for 30 min to stop the tagmentation and then at 70°C for 20 min to inactivate proteinase K. DNA was then extracted with AMPure XP beads (Beckham Counter) and eluted. For PCR, the following thermocycler programme was used: 72°C for 5 min, 98°C for 30 s, 14 cycles of 98°C for 10 s and 63°C for 30 s, final extension at 72°C for 1 min, and hold at 8°C. Pooled libraries were purified with 1.1× AMPure XP beads. Paired‐end Illumina sequencing was carried out on a HiSeq 4000 instrument (Illumina).
8
RNA Extraction and RNA-seq Analysis of Colonoids
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To extract RNA from colonoids, the RNeasy mini kit (#74106, Qiagen, Hilden, Germany) was used as per the manufacturer’s instructions. RNA sequencing (RNA-seq) of colonoids was performed as described before (43 (link)). RNA-seq libraries were generated using SENSE total RNASeq library prep kit with RiboCop rRNA depletion (Lexogen GmbH, Vienna, Austria). Sequencing consisting of 75 single-end reads was performed using Illumina HiSeq4000 instrument (Illumina, Inc., San Diego, CA, USA), and FASTQ files were generated using bcl2fastq 2.18 (Illumina) as per manufacturer’s protocols.
All RNA-seq data analysis was performed in R software. LIMMA linear models with least squares regression and empirical Bayes moderated t statistics were used to identify differential gene expression between experimental groups, and Benjamini–Hochberg false discovery rate (FDR) correction-adjusted P values ≤ 0.05 were considered statistically significant. Enrichment analyses were generated with MetaCore+MetaDrug™ version 21.3 build 7060. Normalized reads and log2 values for all experiments are mentioned inSupplementary File SF2
All RNA-seq data analysis was performed in R software. LIMMA linear models with least squares regression and empirical Bayes moderated t statistics were used to identify differential gene expression between experimental groups, and Benjamini–Hochberg false discovery rate (FDR) correction-adjusted P values ≤ 0.05 were considered statistically significant. Enrichment analyses were generated with MetaCore+MetaDrug™ version 21.3 build 7060. Normalized reads and log2 values for all experiments are mentioned in
9
Exome Capture Sequencing of Conifer Genomes
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Due to the substantial size and complexity of conifer genomes, exome sequence capture is the preferred genotyping method for the taxon (Lind et al., 2022 (link)). We designed our capture probes based on previous Pinus contorta exome capture probes (Suren et al., 2016 (link)) and removed all probes which failed to effectively capture the target sequences. In addition, we included probes corresponding to previously described pathogen response genes (Lu et al., 2021 (link)), filtering out those which targeted exon sequences of less than 100 base pairs (bp). We then submitted our probe sequences to Roche NimbleGen for Custom SeqCapEZ probe design. We achieved a final capture space of ~44 Mbp.
Following DNA extraction and probe design, approximately 100 ng of DNA from each sample was used to construct a barcoded library (Kapa, Dual‐Indexed Adapter Kit). Sequence capture was performed following the SeqCap EZ HyperCap Workflow User's Guide Version 2.0 (Roche Sequencing Solutions, Inc.). The enriched capture libraries were then multiplexed and sequenced in four lanes, with 23–30 libraries per lane, on an Illumina HiSeq4000 instrument at the Centre d'expertise et de services Génome Québec, Montreal, Canada, resulting in 351 Gbp of 150‐bp paired‐end reads.
Following DNA extraction and probe design, approximately 100 ng of DNA from each sample was used to construct a barcoded library (Kapa, Dual‐Indexed Adapter Kit). Sequence capture was performed following the SeqCap EZ HyperCap Workflow User's Guide Version 2.0 (Roche Sequencing Solutions, Inc.). The enriched capture libraries were then multiplexed and sequenced in four lanes, with 23–30 libraries per lane, on an Illumina HiSeq4000 instrument at the Centre d'expertise et de services Génome Québec, Montreal, Canada, resulting in 351 Gbp of 150‐bp paired‐end reads.
10
Transcriptomic Analysis of PM10 Exposure
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Total RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s instructions. RNA concentration/purity were determined by spectrophotometry and agarose gel electrophoresis. For the mRNA sequencing library, six untreated and six PM10 treated samples were constructed with TruSeq Stranded mRNA Sample Prep Kits (Illumina, USA). Samples were indexed with adaptors and submitted for pair-end sequencing using a HiSeq 4000 instrument (Illumina, USA). mRNA libraries were sequenced with a sequencing depth of at least 20 million reads per sample. RNAseq data were analyzed as described before57 (link). Then genes were considered differentially expressed if there was an absolute value of log2 fold change ≥ 1; and the FDR ≤ 0.05. For assessing mRNA transcript abundance, reads were converted to transcripts per million (TPM).
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