tapestation d5000, by Agilent Technologies - Product details

1

Target-Capture Library Preparation and Sequencing

Check if the same lab product or an alternative is used in the 5 most similar protocols

Before continuing we collected the target-capture ‘slurry’ from the previous step, fresh 80% EtOH, DNA purification beads, Equinox Library Amplification Mix 2X, Amplification primers ILMN (Twist Fast Hybridization and Wash Kit with Amp Mix, 104180; Twist Binding and Purification Beads, 100983), a Qubit dsDNA Assay Kit for mass quantification of library, and an Agilent Tapestation D5000 or Agilent BioAnalyzer to calculate molarity under specified regions.
A thermal cycler (lid 105°C) was programmed with the following steps: initialization step 98°C 45s x1, [denature 98°C 15s, anneal 60°C 30s, extend 72°C 30s, x8 cycles], final extension 72°C 1 min, hold 4°C. The slurry was mixed by pipetting. 22.5 μl of the slurry was transferred to a 0.2 mL PCR tube. The rest of the slurry was stored on ice and if not used, frozen for subsequent use. To the tube were added 2.5 μl of Amplification Primers ILMN, and 25 μl of 2X Equinox Amplification Mix for a total of 50 μl. The slurry was amplified in the thermal cycler for 8 cycles. The reaction was bead-cleaned and taken up in 30 μl. 1 μl was quantified by Qubit and 1ng/μl was visualized and quantified on an Agilent Tapestation. The library was sequenced with an Illumina NextSeq 2000 using a P2 200 cycle kit.

Acharya S.N., Nichols R.V., Rylaarsdam L.E., O’Connell B.L., Braun T.P, & Adey A.C. (2023). sciMET-cap: High-throughput single-cell methylation analysis with a reduced sequencing burden. bioRxiv.

+ Open protocol

+ Expand

2

High-Throughput HLA Typing from Blood

Check if the same lab product or an alternative is used in the 5 most similar protocols

High quality DNA was extracted from whole blood aliquots from each participant using the Zymo Quick-DNA 96 Plus Kit (Qiagen). DNA was quantified on the Nanodrop (Thermo Scientific). HLA typing of each participant was performed using the AllType NGS 11-Loci Amplification Kit (One Lambda; Lot 013) according to manufacturer’s instructions. Briefly, 50 ng DNA was amplified using AllType NGS 11-Loci amplification primers. The amplified product was then cleaned and quantified on the Qubit 4.0 (Invitrogen). Library preparation of purified amplicons was carried out as described in the protocol, and the AllType NGS Index Flex Kit (Lot 011) was used for barcoding and secondary amplification. Purified, barcoded libraries were quantified using the Qubit DNA HS kit (Invitrogen) and pooled according to the One Lambda Library Pooling table. Pools of up to 48 libraries were then purified and quantified on the TapeStation D5000 (Agilent) before sequencing on a full MiSeq lane at 150×150bp following manufacturer’s sequencing specifications. HLA types were called using the TypeStream Visual Software from One Lambda. HLA typing results can be found in Supplementary Table 2.

Minervina A.A., Pogorelyy M.V., Kirk A.M., Crawford J.C., Allen E.K., Chou C.H., Mettelman R.C., Allison K.J., Lin C.Y., Brice D.C., Zhu X., Vegesana K., Wu G., Trivedi S., Kottapalli P., Darnell D., McNeely S., Olsen S.R., Schultz-Cherry S., Estepp J.H., McGargill M.A., Wolf J, & Thomas P.G. (2022). SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8 T cells. Nature immunology, 23(5), 781-790.

+ Open protocol

+ Expand

3

Single-cell RNA-seq analysis of MEER cell lines

Check if the same lab product or an alternative is used in the 5 most similar protocols

MEER^vvS and MEER^vvR were cultured in vitro as described above, and cDNA was generated using SMART-seq HT kit (Cat # 634456; Clontech) using 1,000 cells. cDNA product was checked by Tape Station D5000 from Agilent Technologies 2200 to make sure cDNA was successfully generated. Library construction was done using Nextrera XT kit # 15031942 from Illumina. 1 ng cDNA was used in a total volume 5 µl. Sequencing was done using NextSeq 500 System. High Output 75 Cycles kit with run Parameter Paired Read 150 cycles (2 × 75).
Sequencing reads were trimmed for adapters and then aligned to Mus musculus reference genome (Mus_musculus_ensembl_v80_Sequence) using CLC Genomics Workbench. Using CLC Genomics Workbench, differential gene expression was found with statistical cut-offs of P value <0.05, max group means >1, and |foldchange| >1.5. Heatmaps were generated using R package pheatmap with log₂ transformed transcript counts per million (trimmed mean of M-values adjusted).
Gene set enrichment analysis (Mootha et al., 2003 (link); Subramanian et al., 2005 (link)) was performed on differentially expressed genes using the Hallmark gene set.

DePeaux K., Rivadeneira D.B., Lontos K., Dean V.G., Gunn W.G., Watson M.J., Yao T., Wilfahrt D., Hinck C., Wieteska L., Thorne S.H., Hinck A.P, & Delgoffe G.M. (2023). An oncolytic virus–delivered TGFβ inhibitor overcomes the immunosuppressive tumor microenvironment. The Journal of Experimental Medicine, 220(10), e20230053.

+ Open protocol

+ Expand

4

Transcriptome Profiling of Regulatory T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

1000 low glucose and high glucose consuming T_reg cells were sorted directly into lysis buffer in a 96-well plate. Immediately after sorting cDNA was generated using SMART-seq HT kit (Cat # 634456 Clontech) using 1000 cells. cDNA product was checked by Tape Station D5000 from Agilent technologies 2200 to make sure cDNA was successfully generated. Library construction was done using Nextrera XT kit # 15031942 from Illumina. 1ng cDNA was used in total volume 5ul. Sequencing was done using NextSeq 500 System. High Output 75 Cycles kit with run Parameter Paired Read 150 cycles (2X75).
Sequencing reads were trimmed for adapters using Cutadapt⁴³ prior to being aligned to Mus musculus reference genome (mm10) using the RNA-seq aligner HISAT2. Subread’s featureCounts function was used for gene level quantification and results were normalized to Transcripts Per Kilobase Million (TPM). Using the raw quantification, differential genes were found with the R package DESeq2 and statistical cutoffs of q-value <0.05 and |log2foldchange|>1.5.

Watson M.J., Vignali P.D., Mullett S.J., Overacre-Delgoffe A.E., Peralta R.M., Grebinoski S., Menk A.V., Rittenhouse N.L., DePeaux K., Whetstone R.D., Vignali D.A., Hand T.W., Poholek A.C., Morrison B.M., Rothstein J.D., Wendell S.G, & Delgoffe G.M. (2021). Metabolic support of tumor-infiltrating regulatory T cells by lactic acid. Nature, 591(7851), 645-651.

+ Open protocol

+ Expand

5

Transcriptome Profiling of Regulatory T Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

1000 low glucose and high glucose consuming T_reg cells were sorted directly into lysis buffer in a 96-well plate. Immediately after sorting cDNA was generated using SMART-seq HT kit (Cat # 634456 Clontech) using 1000 cells. cDNA product was checked by Tape Station D5000 from Agilent technologies 2200 to make sure cDNA was successfully generated. Library construction was done using Nextrera XT kit # 15031942 from Illumina. 1ng cDNA was used in total volume 5ul. Sequencing was done using NextSeq 500 System. High Output 75 Cycles kit with run Parameter Paired Read 150 cycles (2X75).
Sequencing reads were trimmed for adapters using Cutadapt⁴³ prior to being aligned to Mus musculus reference genome (mm10) using the RNA-seq aligner HISAT2. Subread’s featureCounts function was used for gene level quantification and results were normalized to Transcripts Per Kilobase Million (TPM). Using the raw quantification, differential genes were found with the R package DESeq2 and statistical cutoffs of q-value <0.05 and |log2foldchange|>1.5.

Watson M.J., Vignali P.D., Mullett S.J., Overacre-Delgoffe A.E., Peralta R.M., Grebinoski S., Menk A.V., Rittenhouse N.L., DePeaux K., Whetstone R.D., Vignali D.A., Hand T.W., Poholek A.C., Morrison B.M., Rothstein J.D., Wendell S.G, & Delgoffe G.M. (2021). Metabolic support of tumor-infiltrating regulatory T cells by lactic acid. Nature, 591(7851), 645-651.

+ Open protocol

+ Expand

6

High-throughput HLA Typing from Whole Blood

Check if the same lab product or an alternative is used in the 5 most similar protocols

High quality DNA was extracted from whole blood aliquots from each participant using the Zymo Quick-DNA 96 Plus Kit (Qiagen). DNA was quantified on the Nanodrop (Thermo Scientific). HLA typing of each participant was performed using the AllType NGS 11-Loci Amplification Kit (One Lambda; Lot 013) according to manufacturer’s instructions. Briefly, 50 ng DNA was amplified using AllType NGS 11-Loci amplification primers. The amplified product was then cleaned and quantified on the Qubit 4.0 (Invitrogen). Library preparation of purified amplicons was carried out as described in the protocol, and the AllType NGS Index Flex Kit (Lot 011) was used for barcoding and secondary amplification. Purified, barcoded libraries were quantified using the Qubit DNA HS kit (Invitrogen) and pooled according to the One Lambda Library Pooling table. Pools of up to 48 libraries were then purified and quantified on the TapeStation D5000 (Agilent) before sequencing on a full MiSeq lane at 150×150bp following manufacturer’s sequencing specifications. HLA types were called using the TypeStream Visual Software from One Lambda. HLA typing results can be found in Extended data Table 2.

Minervina A.A., Pogorelyy M.V., Kirk A.M., Crawford J.C., Allen E.K., Chou C.H., Mettelman R.C., Allison K.J., Lin C.Y., Brice D.C., Zhu X., Vegesana K., Wu G., Trivedi S., Kottapalli P., Darnell D., McNeely S., Olsen S.R., Schultz-Cherry S., Estepp J.H., McGargill M.A., Wolf J, & Thomas P.G. (2022). SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8 T cells. medRxiv.

+ Open protocol

+ Expand

7

High-Throughput HLA Typing from Blood

Check if the same lab product or an alternative is used in the 5 most similar protocols

High quality DNA was extracted from whole blood aliquots from each participant using the Zymo Quick-DNA 96 Plus Kit (Qiagen). DNA was quantified on the Nanodrop (Thermo Scientific). HLA typing of each participant was performed using the AllType NGS 11-Loci Amplification Kit (One Lambda; Lot 013) according to manufacturer’s instructions. Briefly, 50 ng DNA was amplified using AllType NGS 11-Loci amplification primers. The amplified product was then cleaned and quantified on the Qubit 4.0 (Invitrogen). Library preparation of purified amplicons was carried out as described in the protocol, and the AllType NGS Index Flex Kit (Lot 011) was used for barcoding and secondary amplification. Purified, barcoded libraries were quantified using the Qubit DNA HS kit (Invitrogen) and pooled according to the One Lambda Library Pooling table. Pools of up to 48 libraries were then purified and quantified on the TapeStation D5000 (Agilent) before sequencing on a full MiSeq lane at 150×150bp following manufacturer’s sequencing specifications. HLA types were called using the TypeStream Visual Software from One Lambda. HLA typing results can be found in Supplementary Table 2.

Minervina A.A., Pogorelyy M.V., Kirk A.M., Crawford J.C., Allen E.K., Chou C.H., Mettelman R.C., Allison K.J., Lin C.Y., Brice D.C., Zhu X., Vegesana K., Wu G., Trivedi S., Kottapalli P., Darnell D., McNeely S., Olsen S.R., Schultz-Cherry S., Estepp J.H., McGargill M.A., Wolf J, & Thomas P.G. (2022). SARS-CoV-2 antigen exposure history shapes phenotypes and specificity of memory CD8 T cells. Nature immunology, 23(5), 781-790.

+ Open protocol

+ Expand

8

Quantification and Sequencing of Final Libraries

Check if the same lab product or an alternative is used in the 5 most similar protocols

Final libraries were quantified using Tapestation D5000 and D1000 reagents on a 2200 TapeStation system (Agilent). Each donor sample was mixed individually based on the molarity of the libraries (45% for each gene expression library, 10% TCR library) and sequenced with 2x150 bp paired-end sequencing on an Illumina HiSeq 4000.

Biermann J., Melms J.C., Amin A.D., Wang Y., Caprio L.A., Karz A., Tagore S., Barrera I., Ibarra-Arellano M.A., Andreatta M., Fullerton B.T., Gretarsson K.H., Sahu V., Mangipudy V.S., Nguyen T.T., Nair A., Rogava M., Ho P., Koch P.D., Banu M., Humala N., Mahajan A., Walsh Z.H., Shah S.B., Vaccaro D.H., Caldwell B., Mu M., Wünnemann F., Chazotte M., Berhe S., Luoma A.M., Driver J., Ingham M., Khan S.A., Rapisuwon S., Slingluff CL J.r., Eigentler T., Röcken M., Carvajal R., Atkins M.B., Davies M.A., Agustinus A., Bakhoum S.F., Azizi E., Siegelin M., Lu C., Carmona S.J., Hibshoosh H., Ribas A., Canoll P., Bruce J.N., Bi W.L., Agrawal P., Schapiro D., Hernando E., Macosko E.Z., Chen F., Schwartz G.K, & Izar B. (2022). Dissecting the treatment-naïve ecosystem of human melanoma brain metastasis. Cell, 185(14), 2591-2608.e30.

+ Open protocol

+ Expand

Tapestation d5000

Lab products found in correlation

8 protocols using tapestation d5000

Target-Capture Library Preparation and Sequencing

High-Throughput HLA Typing from Blood

Single-cell RNA-seq analysis of MEER cell lines

Transcriptome Profiling of Regulatory T Cells

Transcriptome Profiling of Regulatory T Cells

High-throughput HLA Typing from Whole Blood

High-Throughput HLA Typing from Blood

Quantification and Sequencing of Final Libraries

About PubCompare

Ready to get started?