Difference between revisions of "Faq"

Latest revision as of 13:58, 4 June 2020

FAQ's

Frequently Asked Questions

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.

NGS Data Analysis For External Customers

We can provide some analysis results using standardized methods along with the processed data. Please email a brief description of your analysis request to Sumeet Gupta (sgupta@wi.mit.edu) for review. Any additional charges would be communicated on a project by project basis.

@@ Line 9: / Line 9: @@
 == General ==
-=== Which platforms do you support for expression analysis? ===
+=== How do I sign up to use your RT-PCR machine? ===
-We support Affymetrix, Agilent and printed arrays from Operon.
+To sign up for time, please see the calendar - http://genomecore.wi.mit.edu/index.php/Calendar.
-=== How do I sign up to use your ABI Prism 700 RT-PCR machine? ===
+=== Do you have bench/work space for visiting users in the WIGTC laboratory? What about tips, etc? ===
-To sign up for time on the ABI Prism, please see the calendar  on the desk mat underneath the laptop which runs the  machine. Please note that there are time blocks, which are  explained in a note on the wall by the calendar. These time  blocks include only machine time. Please plan accordingly.
-=== Do you have bench/work space for visiting users in the  WICMT 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.
@@ Line 24: / Line 21: @@
 Yes. We will ship your samples and leftover arrays back  to you. Please provide a FedEx number on your sample  submission form.
-=== How long can I expect to wait for my project to be  completed? ===
-We pride ourselves on fast turnaround but our project list  can get quite long. At the time of sample submission we will  provide you with an estimate of when the project will be  completed. Expression analysis projects cannot be scheduled until RNA is submitted. Typical wait time for standard service projects is  about 2 weeks.
 == Pricing ==
-=== How much does it cost to do a microarray experiment? ===
+=== How much will my experiment cost? ===
-Please see the Pricing page for our full price list. Please  note that non-WI/MIT academic institutions should add 30%  to the list price. Commercial entities should add 60%. Contact us for  Affymetrix Gene Chip or Agilent catalog array pricing. Quotes are available for larger projects.   Q: How much does it cost to print a custom array? A: Pricing for custom array projects is a function of labor,  equipment time and supplies (slides). Every project is different  and it is often difficult to predict the total costs until the work  is complete. Customers interested in printing custom arrays  should contact Tom Volkert for estimates.    Q: Is there a bulk discount for processing large numbers of  samples? A: Volume discounts are available, contact WICMT for details.
+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
-=== How much does the lab use program cost? What does it include? ===
-A monthly fee of $250 covers your lab’s usage of  common equipment, such as the Nanodrop spectrophotometer  and the SpeedVac, and occasional usage of other equipment  as needed. Please note that this monthly lab use fee does NOT  include the ABI Prism 700 RT-PCR machine, Affymetrix  equipment, or the Biomek. To sign your lab up, contact  Jennifer Love with your request. You will begin receiving  monthly invoices from WICMT right away.
 == Sample Submission ==
-=== How much RNA do I need to do a microarray experiment? ===
+=== Where can I find the forms I need to submit samples? ===
-For the standard one-cycle labeling protocol we request 10ug  total RNA, and an extra 1ug for QC. For the small  sample (two-cycle) labeling protocol, 25-50ng total RNA is  requested. It is always beneficial to supply enough RNA for  two sample preps, if possible. This is just for security. One and two cycle labeling is available for all supported array platforms.
+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.
-=== How do I submit samples to WICMT for processing? ===
+==== What forms/paperwork should I provide with my sample? ====
-Please download the Sample Submission Form  on the Forms page. There is no need to submit any secondary forms,  since this form provides your contact information, as well as  information about your samples.
+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? ====
+X 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.
-=== Where can I find the forms I need to submit samples? ===
-See our Forms page for all our submission forms.
 == Shipping ==
-=== How do I ship my RNA to WICMT? ===
+=== 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. WICMT will chill your samples overnight to precipitate,  and then spin down and resuspend your RNA. In this case, it is imperative that you supply at least double the amount of RNA  you would normally supply.
+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: Jennifer Love, Whitehead Institute Center for Microarray Technology, 9 Cambridge Center, Room 325, Cambridge MA, 02145.
+Please ship your materials to: Whitehead Institute Genome Technology Core, 455 Main Street, Room 325, Cambridge MA, 02145.
-=== How do I ship my Affymetrix Gene Chips to WICMT? ===
-You can ship the Gene Chips at room temperature by  overnight priority shipping. Once here, they will be stored in  our refrigerators. Please write your name on the boxes before  you send them.
-=== How do I drop-ship Affymetrix Gene Chips to WICMT? ===
+== NGS Data Analysis For External Customers ==
-A: Because WICMT is a service facility, we have made  arrangements with Affymetrix to allow our customers to order  and pay for Gene Chips themselves, and have the Gene Chips  shipped directly to our laboratory. Please be sure to mention  this arrangement when placing your order. Please have the  chips delivered in your name, care of Jennifer Love. This will make keeping track of arriving chips more efficient. Please see  our shipping address above. WICMT cannot order chips for  customers outside of the Whitehead Institute or MIT.
+We can provide some analysis results using standardized methods along with the processed data. Please email a brief description of your analysis request to Sumeet Gupta (sgupta@wi.mit.edu) for review. Any additional charges would be communicated on a project by project basis.