Difference between revisions of "Faq"
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| − | == | + | == Sample Submission == |
| − | === I just want standard RNA-seq data for expression analysis, which kit should I use? === | + | === Where can I find the forms I need to submit samples? === |
| + | See our Forms page for all our submission forms - http://genomecore.wi.mit.edu/index.php/Forms | ||
| + | |||
| + | === 10X Single Cell RNA === | ||
| + | ==== How many samples per lane can I sequence? ==== | ||
| + | We recommend 1 lane of high-output sequencing for every 10,000 cells targeted (You will recover data for about 5000 cells based on what we typically see as a recovery rate). This corresponds to roughly 50K reads per cell. | ||
| + | |||
| + | ==== What sequencing length should I request? ==== | ||
| + | The read length for 10X is standardized and users should request 28x40 read length on the submission form. Longer/custom read lengths are possible in hiseq rapid mode. | ||
| + | |||
| + | ==== What forms/paperwork should I provide with my sample? ==== | ||
| + | Because customers are brining BL2 material into the Whitehead Institute, 10X sample submissions require a cell intake form, in addition to the online submission form. Both forms are available here: http://genomecore.wi.mit.edu/index.php/Forms | ||
| + | |||
| + | ==== What buffers should I use to resuspend my cells on the day of submission? ==== | ||
| + | Here are recommendations from 10x - https://kb.10xgenomics.com/hc/en-us/articles/115001937123-What-buffers-can-be-used-for-washing-and-cell-resuspension- | ||
| + | |||
| + | Full Cell Preparation Guide: https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-single-cell-protocols-cell-preparation-guide#header | ||
| + | |||
| + | ==== What should I do if I have too many dead cells in my suspension? ==== | ||
| + | Dead cells lyse easily and ambient RNA finds its way into GEMs, making data more noisy with unreliable results. Excessive dead cells should be removed prior to submission. | ||
| + | |||
| + | Dead Cell Removal Protocol: https://support.10xgenomics.com/single-cell-vdj/sample-prep/doc/technical-note-removal-of-dead-cells-from-single-cell-suspensions-improves-performance-for-10x-genomics-single-cell-applications#header | ||
| + | |||
| + | ==== Do you count my cells and assess viability prior to sample prep? ==== | ||
| + | No. Due to the high variability in cell size/shape, we rely on the user to be the expert at counting their cell type of interest. Customers should count their cells and measure the viability prior to delivery of samples. | ||
| + | |||
| + | ==== What concentration should I provide my cells at? ==== | ||
| + | Cell concentration is dependent on your target cell number, as the reaction volumes are limiting. | ||
| + | |||
| + | Please refer to the cell suspension chart [need to find a link on 10x website]. Note that the customer should provide cells at the stock concentration listed on the left hand side. PLEASE DO NOT MAKE THE MIX IN THE SQUARES. | ||
| + | |||
| + | ==== Can I provide my cells to you frozen or fixed? ==== | ||
| + | 10X does provide protocols for freezing and fixing cells. However, at this time, the Whitehead Core cannot accept frozen or fixed cells except by special arrangement. We ask that users thaw or rehydrate at their own facility, remove dead cells as necessary, and count prior to delivery. | ||
| + | |||
| + | Frozen cells: https://kb.10xgenomics.com/hc/en-us/articles/360002534012-How-can-I-ship-cells- | ||
| + | Fixed cells: https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-methanol-fixation-of-cells-for-single-cell-rna-sequencing | ||
| + | |||
| + | ==== Can I provide isolated nuclei? ==== | ||
| + | Yes the 3’ single-cell RNA kit works with isolated nuclei: https://support.10xgenomics.com/single-cell-gene-expression/index/doc/demonstrated-protocol-isolation-of-nuclei-for-single-cell-rna-sequencing | ||
| + | |||
| + | ==== What other 10X platforms do you support? ==== | ||
| + | We are willing to work with you on almost any assay the 10X platform offers. 10X currently offers single cell genomics, transcriptomics and epigenomics assays, as well as spatial transcriptomics and linked-reads genomics. For the full list of products, see the 10X website: https://www.10xgenomics.com/ | ||
| + | |||
| + | |||
| + | === Bulk RNA-Seq === | ||
| + | ==== I just want standard RNA-seq data for expression analysis, which kit should I use? ==== | ||
KAPA Hyperprep mRNA if you have more than 100ng of input RNA | KAPA Hyperprep mRNA if you have more than 100ng of input RNA | ||
| − | === I have less than 100ng of input RNA, can I still use KAPA Hyperprep? === | + | ==== I have less than 100ng of input RNA, can I still use KAPA Hyperprep? ==== |
If the RNA is high quality, KAPA HyperPrep may work with less than 100ng but it is risky. A safer approach is to use 10ng in the Clonetech SMART-seq v4 Ultra-low kit. | If the RNA is high quality, KAPA HyperPrep may work with less than 100ng but it is risky. A safer approach is to use 10ng in the Clonetech SMART-seq v4 Ultra-low kit. | ||
| − | === What’s the difference in the data between KAPA HyperPrep and Clonetech Ultralow? === | + | ==== What’s the difference in the data between KAPA HyperPrep and Clonetech Ultralow? ==== |
KAPA data is stranded and probably higher fidelity. The Clonetech Ultralow kit involves more amplification and that can lead to bias but for low input it is the best kit available. | KAPA data is stranded and probably higher fidelity. The Clonetech Ultralow kit involves more amplification and that can lead to bias but for low input it is the best kit available. | ||
| − | === Can I compare data from different kits? === | + | ==== Can I compare data from different kits? ==== |
Kits with similar approaches from different companies will generally be comparable (e.g. KAPA HyperPrep, TruSeq mRNA and NEB Next Ultra II) HOWEVER additional processing/correction may be required. Care should be taken if trying to compare one of the “standard” kits to a low input kit like Clonetech UltraLow due to possible bias in the low input kit. | Kits with similar approaches from different companies will generally be comparable (e.g. KAPA HyperPrep, TruSeq mRNA and NEB Next Ultra II) HOWEVER additional processing/correction may be required. Care should be taken if trying to compare one of the “standard” kits to a low input kit like Clonetech UltraLow due to possible bias in the low input kit. | ||
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Revision as of 11:12, 5 February 2020
|
Contents
- 1 General
- 2 Pricing
- 3 Sample Submission
- 3.1 Where can I find the forms I need to submit samples?
- 3.2 10X Single Cell RNA
- 3.2.1 How many samples per lane can I sequence?
- 3.2.2 What sequencing length should I request?
- 3.2.3 What forms/paperwork should I provide with my sample?
- 3.2.4 What buffers should I use to resuspend my cells on the day of submission?
- 3.2.5 What should I do if I have too many dead cells in my suspension?
- 3.2.6 Do you count my cells and assess viability prior to sample prep?
- 3.2.7 What concentration should I provide my cells at?
- 3.2.8 Can I provide my cells to you frozen or fixed?
- 3.2.9 Can I provide isolated nuclei?
- 3.2.10 What other 10X platforms do you support?
- 3.3 Bulk RNA-Seq
- 4 Shipping
General
How do I sign up to use your RT-PCR machine?
To sign up for time, please see the calendar - http://genomecore.wi.mit.edu/index.php/Calendar.
Do you have bench/work space for visiting users in the WIGTC laboratory? What about tips, etc?
WICMT has available bench space for visitors to use. We supply tips and gloves. Please be aware that this bench space, as well as WICMT refrigerators and freezers, is a “glove zone” – please wear gloves when handling WICMT tips, reagents, etc.
How is my RNA checked for quality control before processing?
We use Agilent’s Bioanalyzer 2100 for RNA QC. Please see Agilent’s website for more information.
Will I get my samples back after my experiment is complete?
Yes. We will ship your samples and leftover arrays back to you. Please provide a FedEx number on your sample submission form.
Pricing
How much will my experiment cost?
Please see the Pricing page for our full price list. Every project is different and it is often difficult to predict the total costs until the work is complete. Customers interested in quotes - please email wibr-genome@wi.mit.edu
Sample Submission
Where can I find the forms I need to submit samples?
See our Forms page for all our submission forms - http://genomecore.wi.mit.edu/index.php/Forms
10X Single Cell RNA
How many samples per lane can I sequence?
We recommend 1 lane of high-output sequencing for every 10,000 cells targeted (You will recover data for about 5000 cells based on what we typically see as a recovery rate). This corresponds to roughly 50K reads per cell.
What sequencing length should I request?
The read length for 10X is standardized and users should request 28x40 read length on the submission form. Longer/custom read lengths are possible in hiseq rapid mode.
What forms/paperwork should I provide with my sample?
Because customers are brining BL2 material into the Whitehead Institute, 10X sample submissions require a cell intake form, in addition to the online submission form. Both forms are available here: http://genomecore.wi.mit.edu/index.php/Forms
What buffers should I use to resuspend my cells on the day of submission?
Here are recommendations from 10x - https://kb.10xgenomics.com/hc/en-us/articles/115001937123-What-buffers-can-be-used-for-washing-and-cell-resuspension-
Full Cell Preparation Guide: https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-single-cell-protocols-cell-preparation-guide#header
What should I do if I have too many dead cells in my suspension?
Dead cells lyse easily and ambient RNA finds its way into GEMs, making data more noisy with unreliable results. Excessive dead cells should be removed prior to submission.
Dead Cell Removal Protocol: https://support.10xgenomics.com/single-cell-vdj/sample-prep/doc/technical-note-removal-of-dead-cells-from-single-cell-suspensions-improves-performance-for-10x-genomics-single-cell-applications#header
Do you count my cells and assess viability prior to sample prep?
No. Due to the high variability in cell size/shape, we rely on the user to be the expert at counting their cell type of interest. Customers should count their cells and measure the viability prior to delivery of samples.
What concentration should I provide my cells at?
Cell concentration is dependent on your target cell number, as the reaction volumes are limiting.
Please refer to the cell suspension chart [need to find a link on 10x website]. Note that the customer should provide cells at the stock concentration listed on the left hand side. PLEASE DO NOT MAKE THE MIX IN THE SQUARES.
Can I provide my cells to you frozen or fixed?
10X does provide protocols for freezing and fixing cells. However, at this time, the Whitehead Core cannot accept frozen or fixed cells except by special arrangement. We ask that users thaw or rehydrate at their own facility, remove dead cells as necessary, and count prior to delivery.
Frozen cells: https://kb.10xgenomics.com/hc/en-us/articles/360002534012-How-can-I-ship-cells- Fixed cells: https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-methanol-fixation-of-cells-for-single-cell-rna-sequencing
Can I provide isolated nuclei?
Yes the 3’ single-cell RNA kit works with isolated nuclei: https://support.10xgenomics.com/single-cell-gene-expression/index/doc/demonstrated-protocol-isolation-of-nuclei-for-single-cell-rna-sequencing
What other 10X platforms do you support?
We are willing to work with you on almost any assay the 10X platform offers. 10X currently offers single cell genomics, transcriptomics and epigenomics assays, as well as spatial transcriptomics and linked-reads genomics. For the full list of products, see the 10X website: https://www.10xgenomics.com/
Bulk RNA-Seq
I just want standard RNA-seq data for expression analysis, which kit should I use?
KAPA Hyperprep mRNA if you have more than 100ng of input RNA
I have less than 100ng of input RNA, can I still use KAPA Hyperprep?
If the RNA is high quality, KAPA HyperPrep may work with less than 100ng but it is risky. A safer approach is to use 10ng in the Clonetech SMART-seq v4 Ultra-low kit.
What’s the difference in the data between KAPA HyperPrep and Clonetech Ultralow?
KAPA data is stranded and probably higher fidelity. The Clonetech Ultralow kit involves more amplification and that can lead to bias but for low input it is the best kit available.
Can I compare data from different kits?
Kits with similar approaches from different companies will generally be comparable (e.g. KAPA HyperPrep, TruSeq mRNA and NEB Next Ultra II) HOWEVER additional processing/correction may be required. Care should be taken if trying to compare one of the “standard” kits to a low input kit like Clonetech UltraLow due to possible bias in the low input kit.
Shipping
How do I ship my RNA to WIGTC?
If you are shipping within the U.S., please send your samples by overnight priority shipping on dry ice. If you are shipping internationally, please send your samples at room temperature, precipitated in EtOH and NaOAc according to your lab protocol.
What’s your shipping address?
Please ship your materials to: Whitehead Institute Genome Technology Core, 455 Main Street, Room 325, Cambridge MA, 02145.
