Genomic DNA was purified using a DNeasy Blood and Tissue kit (Qiagen) and was then fragmented on a Covaris M220 instrument according to the manufacturer’s protocol. Libraries were prepared using Swift S2 Acel reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 200 ng of DNA with 14 cycles of PCR amplification. DNA libraries were quantified on a Qubit 2.0 Fluorometer (Life Technologies) and fragment size distributions were evaluated on a Agilent TapeStation 2200 (Agilent Technologies). Pooled libraries were further evaluated with low-pass sequencing on an Illumina MiSeq and then sequenced to approximately 5× mean genome coverage on an NovaSeq 6000 instrument (Illumina) with 2× 150 bp paired-end configuration in the Molecular Biology Core Facilities at Dana-Farber Cancer Institute. Haplotype-specific DNA copy number was calculated using the same workflow as previously described11 ,48 (link). DNA rearrangements shown in Extended Data Fig. 11 were taken from previous analyses11 .
Biomek i7
Manufactured by Beckman Coulter
Sourced in United States
The Biomek i7 is a liquid handling workstation designed for automated sample processing. It features liquid handling capabilities and can be configured to perform a variety of laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (17 mentions)
Tapestation 4200, by Agilent Technologies (9 mentions)
Kapa mrna hyperprep strand specific sample preparation kit, by Beckman Coulter (6 mentions)
Qubit fluorometer, by Agilent Technologies (6 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Agilent 4200 tapestation, by Agilent Technologies (3 mentions)
M220 instrument, by Covaris (2 mentions)
Rneasy micro mini plus kit, by Qiagen (2 mentions)
Rlt plus lysis buffer, by Qiagen (2 mentions)
Rnadvance viral kit, by Beckman Coulter (2 mentions)
Sensifast probe lo rox one step kit, by Meridian Bioscience (2 mentions)
Trizol plus rna purification kit, by Thermo Fisher Scientific (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Allprep dna rna mirna universal kit, by Qiagen (2 mentions)
Agilent 2200 tapestation, by Agilent Technologies (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Library quantification kit, by Roche (1 mentions)
Taqpath covid 19 rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
29 protocols using biomek i7
1
Genome-wide DNA Copy Number Analysis
2
SARS-CoV-2 Whole Genome Sequencing from Swabs
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Total RNA extracted from swab samples was used for whole genome sequencing of SARS-CoV-2 as previously described6 (link),22 (link)–25 and the sequencing libraries were prepared according to version 4.1 of the ARTIC nCoV-2019 protocol26 . We used a semi-automated workflow that employed BioMek i7 liquid-handling workstations (Beckman Coulter Life Sciences) and MANTIS automated liquid handlers (FORMULATRIX). Using a NovaSeq 6000 instrument (Illumina) we generated short sequence reads to ensure a very high depth of coverage. Sequencing libraries were prepared in duplicate and sequenced with an SP 300 cycle reagent kit.
3
RNA-seq Analysis of DS1 and DS2U hiAS
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Two biological replicates of DS1 and two biological replicates DS2U hiAs were harvested in RLTplus Lysis buffer (Qiagen, 1053393). Total RNA was isolated using the RNeasy micro/mini plus kit (Qiagen, 74034). Libraries were prepared using Roche Kapa mRNA HyperPrep strand specific sample preparation kits from 200ng of purified total RNA according to the manufacturer’s protocol using a Beckman Coulter Biomek i7. The finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in equimolar ratio and subjected to shallow sequencing on an Illumina MiSeq to evaluate library quality and pooling balance. The final pool was sequenced on an Illumina NovaSeq 6000 targeting 30 million 100bp read pairs per library. Sequenced reads were aligned to the UCSC hg19 reference genome assembly and gene counts were quantified using STAR (v2.7.3a).52 (link) Differential gene expression testing was performed by DESeq2 (v1.22.1).54 (link) RNAseq analysis was performed using the VIPER snakemake pipeline.55 (link) Library preparation, Illumina sequencing and VIPER workflow were performed by the Dana-Farber Cancer Institute Molecular Biology Core Facilities. Gene Ontology (GO) analysis of differentially expressed genes was performed using Metascape.56 (link)
4
SARS-CoV-2 Viral Load Quantification
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Viral load in lungs of SARS-CoV-2 infected mice was quantified by qRT-PCR and by plaque assay [4 (link)]. Lungs were ground in 1.5 mL of PBS and 200 µL was added to LBF lysis buffer. RNA was extracted using RNAdvance Viral Kit on a Biomek i7 automated workstation (Beckman Coulter, Indianapolis, IN, USA), according to the manufacturer’s protocol. Each sample was eluted in 50 µL of RNase-free water. RT-PCR was performed using the SensiFASTTM Probe Lo-ROX One-Step kit (Bioline, London, UK). Primers and probe sequences, targeting the SARS-CoV-2 E gene, were based on the Berlin protocol published in the WHO recommendation for the detection of SARS-CoV-2 and as described before [4 (link)]. The thermal cycling reaction was performed at 48 °C for 20 min for reverse transcription, followed by 95 °C for 2 min, and then 45 cycles of 15 s at 94 °C; 35 s at 60 °C for the E gene amplification. Cycle Threshold (Ct) values were converted to PFU equivalents (PFU Eqv.), according to a calibration curve determined in parallel.
5
Comprehensive RNA-Seq Analysis Workflow
6
SARS-CoV-2 Detection by qRT-PCR
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Real-time quantitative reverse transcriptase polymerase chain reactions (qRT-PCRs) were performed using the TaqPath Covid-19 RT-PCR Kit (A48067; ThermoFisher Scientific, Massachusetts, USA), a fast, highly sensitive, multiplex and robust RT-qPCR assay for the detection of SARS-CoV-228 (link). Nucleic acids were isolated according to manufacturer’s instructions. Briefly, 200 μl viral transport medium (VTM) was taken from the patient swab sample inside a class 2 safety cabinet and mixed with 150 μl lysis buffer, 1 μl carrier RNA, and extraction controls (MS2, was provided as part of the kit). After incubation at room temperature for at least 15 min, samples were processed using the liquid handler Biomek i7 automated workstation (Beckman, Coulter) for RNA isolation. Primers and probes to target the SARS-CoV-2 E, N (N1 and N2 targets), and S genes, were included in the kit. A positive result for SARS-CoV-2 detection was determined by amplification of at least two of the three genes targeted, using a cutoff threshold cycle (CT) value of 37.
7
Polysome Profiling of BRD9 Modulation
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Total RNA was extracted from OPM2 and H929 cells using the RNeasy Plus Mini Kit (Qiagen) after 24 hours of treatment with 100 nmol/L of dBRD9-A or DMSO, or after 48 hours of puromycin selection following shBRD9 or shLuc transduction in biological triplicate. For polysome-seq, all polysome (≥3 ribosomes) fractions were pooled, and total RNA was extracted from the same volume of each sample using TRizol Plus RNA Purification Kit (Thermo Fisher Scientific) in biological triplicate. Library preparation was performed in the Molecular Biology Core Facilities at the Dana-Farber Cancer Institute using Roche Kapa mRNA HyperPrep strand-specific sample preparation reagents from 200 ng of purified total RNA on a Beckman Coulter Biomek i7. The finished dsDNA libraries were quantified using the Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual-indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to evaluate library quality and pool balance. The final pool was sequenced on an Illumina NovaSeq 6000 with paired-end 100bp reads at the Dana-Farber Cancer Institute Molecular Biology Core Facilities. RNA-seq and polysome-seq data are in the Gene Expression Omnibus (GEO) database (GSE197487).
8
CX3CL1 Stimulation of Tumor Cells
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Tumor cells were treated with CX3CL1 (Novus Biologicals; cat NBP2-35038) at 2 µg/ml for 8 hours and RNA was prepared using RNAeasy kit (Qiagen Catalog 74104). Following incubation, total RNA was prepared using RNAeasy kit (Qiagen Catalog 74104).
RNA sequencing was performed at the Molecular Biology Core Facility, Dana-Farber Cancer Institute. Libraries were prepared using Roche Kapa mRNA HyperPrep strand specific sample preparation kits from 200ng of purified total RNA (tumor sample) or Takara SmartSeq v4 reagents from 1ng of RNA (MDSC sample) according to the manufacturer’s protocol on a Beckman Coulter Biomek i7. The finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced on an Illumina NovaSeq 6000 targeting 40 million 150bp read pairs per library at the Dana-Farber Cancer Institute Molecular Biology Core Facilities.
RNA sequencing was performed at the Molecular Biology Core Facility, Dana-Farber Cancer Institute. Libraries were prepared using Roche Kapa mRNA HyperPrep strand specific sample preparation kits from 200ng of purified total RNA (tumor sample) or Takara SmartSeq v4 reagents from 1ng of RNA (MDSC sample) according to the manufacturer’s protocol on a Beckman Coulter Biomek i7. The finished dsDNA libraries were quantified by Qubit fluorometer and Agilent TapeStation 4200. Uniquely dual indexed libraries were pooled in an equimolar ratio and shallowly sequenced on an Illumina MiSeq to further evaluate library quality and pool balance. The final pool was sequenced on an Illumina NovaSeq 6000 targeting 40 million 150bp read pairs per library at the Dana-Farber Cancer Institute Molecular Biology Core Facilities.
9
RNA-seq Analysis of DKO and TKO B-ALL Cells
10
High-throughput Screening of Yeast Strains
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The various single clones picked from agar plates were cultured and preserved in 96-well plates, and then the Biomek i7 (Beckman Ltd., USA) automated workstation was used to complete the storage of multiple copies of the sample. In the MicroScreen system (Gering Ltd., China), the growth of Y. lipolytica strains was examined. We transferred 10 μL of the bacterial liquid preserved in the 96-well plate to a seed 48-well plate with 1 mL of 1/4 Delft medium in each well and cultured it for 24–36 h. Thereafter, starting with an initial OD600 of 0.2, an appropriate volume of bacterial liquid from the seed 48-well plate was transferred to a fermentation 48-well plate for further cultivation (each strain has 4 repetitions). Then the accumulation of extracellular total protein was determined by Bradford assay [15 (link)]. The preliminarily screened strains were subjected to re-screening in 250 mL shake flasks at 25 ℃.
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