Chromatin from synchronous-rings-stage parasites of 3D7 clone G7 was prepared and 3*108 cells per ChIP used for the previously described protocol (Lopez-Rubio et al., 2013 (link)). Briefly, chromatin was crosslinked in 1% formaldehyde for 10 min (Sigma-Aldrich, #SZBD1830V), sheared to an average length of 300 bp using the BioRuptor Pico, and individual histone modifications were pulled down using 0.5 μg of antibody for H3K4me3 (Diagenode, cat # K2921004), H3K9me3 (Millipore, cat # 257833), and homemade rabbit polyclonal anti-PfHP1. 5 μl rabbit polyclonal anti-H4K31me1 and 15 μl anti-H4K31ac were used for each experiment. To generate Illumina-compatible sequencing libraries, the immunoprecipitated DNA and input was processed using the MicroPlex Library Preparation Kit (Diagenode C05010014) according to manufacturer’s instructions. The optimized library amplification step used KAPA Biosystems HIFI polymerase (KAPA Biosystems KK2101). Pooled, multiplexed libraries were sequenced on an Illumina NextSeq 500/550 system as a 150-nucleotide single-end run. The raw data were demultiplexed using bcl2fastq2 (Illumina) and converted to fastq format files for downstream analysis. Two biological replicates were analyzed for each antibody.
Nextseq 500 550 system
Manufactured by Illumina
Sourced in United States
The NextSeq 500/550 system is a high-throughput sequencing platform designed for a wide range of applications, including whole-genome sequencing, targeted gene panels, and gene expression analysis. The system utilizes a proprietary sequencing-by-synthesis chemistry to deliver accurate and reliable DNA sequencing results.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500 550 high output kit v2 75 cycle, by Illumina (2 mentions)
Sense total rna seq library prep kit, by Lexogen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
H3k9me3, by Merck Group (1 mentions)
Bcl2fastq2, by Illumina (1 mentions)
Microplex library preparation kit, by Diagenode (1 mentions)
Hifi polymerase, by Roche (1 mentions)
Qubit dsdna broad range assay, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Truseq stranded total rna lt sample prep kit, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Random hexamer, by Thermo Fisher Scientific (1 mentions)
Qubit instrument, by Thermo Fisher Scientific (1 mentions)
Monarch dna clean up kits, by New England Biolabs (1 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Nebnext ultra 2 nondirectional second strand synthesis module, by New England Biolabs (1 mentions)
Nebnext rrna depletion kit human mouse rat, by Illumina (1 mentions)
9 protocols using nextseq 500 550 system
1
Chromatin Immunoprecipitation (ChIP) Protocol for Malaria Parasites
2
Targeted Myeloid Neoplasm Panel Sequencing
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DNA libraries were prepared using the unique molecular identifier (UMI)-based QIAseq Targeted Human Myeloid Neoplasms Panel (Qiagen) and sequenced on an Illumina NextSeq 500/550 system. The panel covers the complete coding region of 141 genes. The read processing, alignment (hg19 as the reference), calling, and annotation of single nucleotide variants/small indels were performed with the UMI-based caller smCounter268 (link). The mean read depth of the target regions across all samples was 1119×.
3
Whole-Genome Resequencing of Evolved B. subtilis
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Start clones and selected evolved isolates of each TALE and ALE experiment were selected and prepared for whole-genome re-sequencing. Genomic DNA was extracted from overnight cultures (grown in LB medium) using the MasterPure Gram Positive DNA Purification Kit (Lucigen). Quality was assessed by evaluating Abs260nm Abs280nm−1 using a Nanodrop (Thermo Fisher scientific, USA). DNA concentration was measured using a Qubit ds-DNA broad range assay (Thermo Fisher scientific, USA), and paired-end sequencing libraries were generated using the Illumina 300 cycle (150 bp x 2) kit (San Diego, CA, USA). Sequencing was performed on an Illumina NextSeq 500/550 system (Illumina, USA). The average coverage for each sample was over 60. Genome sequencing reads were analyzed using the in-house mutation calling pipeline called “ALE mut pipeline” to generate lists of mutations for each evolved strain (Phaneuf et al., 2019a (link)). The reference strain used for this analysis was B. subtilis 168 with the GenBank accession number NC_000964 and B. subtilis PY79 with the GenBank accession number CP006881.1 . Start strain-specific mutations present before evolution were excluded from the analysis.
4
High-Quality RNA Sequencing Protocol
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RNA samples (concentration >210 ng/µL, RNA integrity number >9.0) were used for RNA libraries preparation using [(Poly(A)RNA Selection, Lexogen Sense Total RNA-Seq Library Prep Kit (Lexogen GmbH, Vienna, Austria)]. For sequencing, NextSeq®500/550 High Output Kit v2 (75 cycle) and NextSeq 500/550 system (Illumina, Inc., San Diego, CA, USA) were used. The reactions were performed according to the manufacturers’ recommendations.
5
Next-Gen RNA-Seq Library Prep
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Next-generation sequencing library preparation was performed using the NEXTflex Small RNA-Seq Kit V3 following step A to step G of Bioo Scientific’s standard protocol. Amplified libraries were purified by running an 8% TBE gel and size-selected for 15–40 nucleotides. Libraries were profiled using a High Sensitivity DNA Chip on the 2100 Bioanalyzer (Agilent Technologies), quantified using the Qubit dsDNA HS Assay Kit in the Qubit 2.0 Fluorometer (Life Technologies) and sequenced on the Illumina NextSeq 500/550 system.
6
RNA-seq Analysis of Cell Transcriptome
7
High-Quality RNA Sequencing Library Prep
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RNA samples (concentration > 260 ng/µl, RNA integrity number > 7.5) were used for RNA libraries preparation using (Poly(A)RNA Selection, Lexogen Sense Total RNA-Seq Library Prep Kit (Lexogen GmbH; Vienna, Austria). For sequencing, NextSeq® 500/550 High Output Kit v2 (75 cycle) and NextSeq 500/550 system (Illumina, Inc.; San Diego, CA, USA) were used. The reactions were performed according to the manufacturers’ recommendations.
8
RNA-Seq Library Preparation Protocol
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In vitro-purified RNA (1 μg) was reverse transcribed by using 200 U SuperScript II under standard buffer conditions (50 mM Tris-HCl [pH 8.3], 75 mM KCl, 10 mM dithiothreitol [DTT], 6 mM MnCl2, and 0.5 mM deoxynucleoside triphosphate [dNTP] mix) with random hexamers (LifeTech). Second strand synthesis was performed by using NEBNext Ultra II nondirectional second strand synthesis module (New England BioLabs [NEB]). Double-stranded cDNA was purified with Monarch DNA cleanup kits (NEB) and diluted to 0.2 ng/μL. Sequencing libraries were generated by using a Nextera XT DNA library preparation kit (Illumina) according to the manufacturer’s instructions. Libraries were quantified by using a Qubit instrument (Life Technologies) and bioanalyzer (Agilent) and sequenced on a NextSeq 500/550 system (Illumina).
9
Transcriptome analysis from total RNA
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Total RNA was isolated from 250 µl of lysate with 750 µl of TRIzol LS Reagent and treated with RQ1 RNase-Free DNase (1 Unit for 1 µg of total RNA) for 30 minutes at 37°C with subsequent water saturated acidic phenol extraction and precipitation with ethanol (with the addition of 1/100 volume of glycogen RNA grade). 500 ng of DNase I treated total RNA was depleted of ribosomal RNA with NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (#E6310) and used for transcriptome library preparation with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (#E7760). Both ribosome profiling and transcriptome libraries were sequenced on the Illumina NextSeq 500/550 system (Genome Sequencing Research and Education Center, Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University).
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