Point people/organizer: Yarui Diao

4DN Google Drive folder: https://drive.google.com/drive/folders/12hlDOCN-ulRuri0N4SvjpoWPNJkTdMuW?usp=sharing

4DN Calendar: https://wiki.4dnucleome.org/4dn:calendar_4dn (click here to add this specific WG calendar to your personal calendar)

Agenda/Minutes: https://docs.google.com/document/d/1mXOdciJRXxQXntpKQjS0u-NKYnCm0k-CnsTUkpZe3tI/edit?usp=sharing

Meeting attendee spreadsheet: https://docs.google.com/spreadsheets/d/1zRkKpOaRzJoTg3BZG9Gqs3TiTsLxS5rZsat2Jhk0ts4/edit?usp=sharing

Email list: cells@4dnucleome.org. Emails sent there will go to everyone on the list. Replying to an email from the list will also send it to everyone in the list.

Slack channel: #wg-cells

Cells WG member spreadsheet

One major goal of the 4D Nucleome Network is the integration of a large array of different technologies to study the structure and dynamics of the human genome. The application of these methods to common cells systems using common protocols will greatly facilitate achieving this goal. This working group’s job is to define these protocols for those groups that are working with common systems and for the common systems that will be employed for the 4DN Phase 2 collaborative projects. While individual laboratories will also use lab-specific materials selected for their specific project or technology, wherever possible groups are encouraged to use 4DN Cells WG approved protocols in order for their datasets to be labeled as gold standard for data integration.

4DN Cells Legacy Document - this document contains a summary of the issues faced and decisions made by the Cells WG in 4DN Phase 1

1) To answer queries, update protocols, negotiate standard lots of reagents for the 4DN Tier1 and Tier2 cell line in order to define gold standard datasets for data integration. 2) To identify 4DN groups working with common cell systems, and encourage them to work with the Cells WG to establish common protocols that will permit generation of 4DN gold standard datasets for integrative analyses. 3) To establish standard protocols for stocking and maintaining cell samples for the 4DN collaborative projects and establish the metadata requirements for datasets to be designated gold standard datasets for integrative analyses. 5) To establish protocols to engineer and differentiate 4DN approved cell lines and make 4DN members aware of these cell lines to avoid duplication of effort.

The mailing list for the working group has been changed. Please use cells@4dnucleome.org from now on as the new mailing list address.

IF YOU ARE HAVING TROUBLE VIEWING ANY OF THE LINKS IN THIS SITE READ THIS:

This is most likely caused by having multiple Google accounts open in the same browser (probably across multiple tabs). Some of the documents on 4DN Wiki have a sharing setting that it will open only if the “active” Google account is the one you are authorized to access 4DN Wiki. To see your “active” Google account, move your mouse within the page until the top bar appears.

To see the document, you may need to

• Log off your other Google account(s) in other Google pages until the one you use for 4DN Wiki becomes active, then refresh the document page, or

• Log off all your Google accounts, refresh the document page, the avatar will be replaced by a “Sign in” button which you may click to use your 4DN account to sign in

You can find this information in the 4DN wiki > community information > Question 19 on the following page: community_information

Chair:

Yarui Diao, yarui.diao@duke.edu

Regular Cells WG meetings will be held on 3rd Fridays of every month.

ZOOM CONTACT INFORMATION:

Join Zoom Meeting
https://4dnucleome-org.zoom.us/j/94419417579?pwd=aWFCbWxvVzY3R0RzOEU3a0RYWTRFdz09

Meeting ID: 944 1941 7579
Passcode: 985933

KATE RIVERA-NOEL (oh@4dnucleome.org)

BELOW ARE THE INSTRUCTIONS FOR STOCKING, EXPANDING 4DN TIER1 AND TIER2 CELL LINES INCLUDING REQUIRED SOPS, REAGENT LOT NUMBERS TO GENERATE 4DN GOLD STANDARD DATASETS WITH THESE CELL LINES.

INSTRUCTIONS FOR OBTAINING CELL LINES, INCLUDING MTAs, ARE FOUND ON THE CELL REPOSITORY WIKI PAGE

Tier 1 Lines - CLICK ON LINE FOR THE LATEST 4DN GROWTH SOP NECESSARY FOR GOLD STANDARD DATA PRODUCTION!!! NOTE: For cell lines that use FBS, use the negotiated 4DN lot of FBS. Brief 4DN history of Tier1 lines is provided. The top priorities for Tier1 were that they have a (relatively) stable karyotype and can be expandable to large numbers. Secondary issues that excluded lines from Tier1 were onerous MTAs or licences (e.g. Geron hTERT plasmid construct in RPE), derivation from tumors (e.g. HCT116), lack of consent for public sharing of raw DNA sequence reads and mouse cells. 4DN Cells WG is always willing to consider any changes or any additions to Tiered cells line (Tier1 or Tier2).

H1 male hESC line

WA01 (aka H1) was chosen due to its abundance of genomics data. Human ESCs offer differentiation toward many different cell lineages. Cells are immortal, and methods for genome engineering are continuously improving. Both fixed and live imaging have been achieved and the motility of the cells has been addressed with cell density and ROCK inhibition. One disadvantage is that they are more challenging to grow and particularly to maintain in an undifferentiated state but in some cases experienced labs could provide fixed material to less experienced labs.

GM12878

(*) IMR90 (note particularly restrictive MTA terms)

IMR90 and GM12878. Many investigators feel strongly about these cell lines due to the amount of data available in the worldwide scientific community and the momentum that they have accrued. Disadvantages are that IMR90 is not immortal and may change with passage and will eventually undergo growth senescence, while GM12878 is very difficult to transfect and is a suspension cell line that poses challenges for imaging methods. However, these cell lines will serve a large number of 4DN labs wanting to do big data enabled computational modeling.

(*) HFFc6

hTERT-HFFc6. Many 4DN imaging applications require flat, adherent cells. hTERT-immortalized HFF cells remain diploid over multiple passages (according to users; no data as yet provided) and garnered the most enthusiasm for this purpose from the feedback we received. Since the most commonly used hTERT plasmid is marketed by Geron and imposes a restrictive licence, hTERT-HFF cell line was immortalized using a modified hTERT construct that is not subject to the Geron licence.

WTC-11 - obtain from CORIELL

WTC-11 is an iPSC cell line that is fully consented for public sharing of sequencing data, differentiates well to various lineages, exhibits a stable karyotype, and is amenable to gene editing. Allen Institute for Cell Science has used WTC-11 exclusively in its development of myriad GFP fusion protein expressing lines and establishment of live cell imaging methodologies.

SPECIAL NOTES ON WTC-11:

Both the parental WTC-11 cell line as well as the gene-edited WTC-11 cells lines generated by the Allen Institute for Cell Science (AICS) can be obtained from Coriell. Please use the AICS’s cell culture SOP to culture both the parental and the cell lines in the AICS Cell Collection. The AICS does not recommend thawing the parental WTC-11 cell line using the thawing step outlined in their SOP. Due to slight differences in the banking approach of the parental cell line, they recommend using the thawing step of Coriell’s WTC-11 SOP for optimal post-thaw recovery of the parental WTC-11 cell line. The AICS’s cell culture SOP is periodically updated so always use the link to see the most recent SOP.Please carefully read “supplementary information” as well!!!

IMPORTANTLY: There are several versions of this cell line available. Which version you purchase will depend on what you intend to do, but all use the same cell culture SOP. What follows are general explanations of what is available in the Coriell catalog to assist you in making your choice.

WTC-11 cat# GM25256: This is the parental “wild-type” cell line. For most engineering purposes, you will want to start with this cell line. Since this line was deposited to Coriell by the Gladstone Institute instead of the AICS, WTC-11’s culture SOP found at Coriell is quite different from other engineered WTC-11 deposited by AICS. For 4DN “gold standard” datasets, please use AICS’ cell culture SOP for passaging the cells even if you use the parental “wild-type” WTC-11 (GM25256).

AICS-0036-006: This cell line expresses cytoplasmic GFP from one allele of the AAVS1 safe harbor and was subjected to the same transfection and expansion process as the other gene-edited cell lines in the AICS Cell Collection. A set of recurrent transcriptome changes were found in these gene-edited cell lines compared to the WTC-11 parental line available on Coriell. These transcriptome changes were traced to passage number. Therefore, some investigators prefer to use AICS-0036-006 as a control.

AICS FP- fusion protein expressing cell lines (cat# varies): The AICS Cell Collection includes many WTC-11-derived cell lines expressing FP-fusion proteins. The catalog of these lines and their Coriell catalog numbers can be found here (AICS cell catalog ).

Tier 2 Lines - CLICK ON LINE FOR THE LATEST 4DN GROWTH SOP!! NOTE: For cell lines that use FBS, use the negotiated 4DN lot of FBS.

F121-9 female CASTx129 hybrid mouse ESCs - obtain from Dave Gilbert.

F123 male CASTx129 hybrid mouse ESCs - obtain SOP and cell line from Bing Ren

K562 - obtain from ATCC

H9 female hESC line - obtain from Wicell and use same SOP as H1

(*) hTERT-RPE - obtain from ATCC (note particularly restrictive MTA terms)

HEK293 - obtain from ATCC

HAP1 - obtain from van Steensel lab (note particularly restrictive MTA terms)

(*) U2OS - obtain from ATCC

(*) HCT116 - obtain from ATCC use modified SOP linked here

(*) JM8.N4 - mouse ESCs good for imaging, obtain cell line from KOMP, follow SOP linked here, imagers are primarily using serum/LIF recipe

G4 - mouse ESCs that have been extensively engineered by the Ritland/Groudine group. Obtain from Carol Ware: cware@u.washington.edu

(*) These lines are recommended for use in imaging by the Imaging Data Standards Working Group

How to obtain 4DN Cell lines

Please refer to the 4DN Cell Repository wiki page

Posting of public data from specimens collected before January 15, 2015 is the responsibility of and must be approved by each PIs institution. Additional questions about Consent should be addressed by individual PIs with their institutions.

NIH Genomic Data Sharing Policy

(EXAMPLE) Florida State University RESEARCH COMPLIANCE page

MTAs are the responsibility of each PI and their institution and are to be negotiated separately between each institution and the distributor. Additional questions about MTAs should be addressed by individual PIs with their institutions. The document below is a list of salient points from the MTAs between the OH (UCSD) and the three major providers, provided to 4DN PIs for your convenience as a courtesy and not to be considered a binding document.

Readers Digest List of MTA Conditions Condensed by the OH

Are You Engineering or Have you Engineered Any of the 4DN Cell Lines or Would You Like to Know What Engineered Lines Have Been or are Being Made?

START by looking at this spread sheet of completed and planned cell lines: Engineered Cell Lines

IF YOU ARE PLANNING TO OR ARE IN THE PROCESS OF MAKING A LINE: Please let the cells working group know (contacts at the top of the page) and please discuss your plans on this discussion group of who is engineering what so as not to duplicate effort or to work together! If you cannot figure out how to post to the forum, we will start it for you when you email us.

IF YOU HAVE AN ENGINEERED LINE YOU ARE WILLING TO SHARE: please let the cells working group know (contacts at the top of this page) and we will post it to the spread sheet above.

These are suggested protocols based on first hand experience from 4DN members

High Efficiency Transfection of hESC line H9 (Note: H1 requires a different protocol)

High Efficiency Transfection and CRISPR editing of F121-9

CRISPR/Cas9 Gene Editing in Human Pluripotent Cell Lines

DATASETS GENERATED USING THE APPROVED SOP PRECISELY AND PROVIDING THE REQUISITE METADATA WILL BE MARKED “GOLD STANDARD” - IF YOU DO NOT FOLLOW THESE PROTOCOLS STRICTLY THEN YOUR DATASETS WILL NOT BE DESIGNATED “GOLD STANDARD”. MAKE SURE TO CHECK THE CELLS WIKI PAGE EACH TIME YOU START AN EXPERIMENT FOR ANY UPDATES ON REAGENTS, ETC.!!

Upon submission of data to the DCC, PIs will be asked to authenticate the cellular material from which data were derived, and whether the handling of the cells deviated from the SOPs provided below, which are intended to minimize variability between labs. Authentication criteria for each cell line may differ. THE SOP FOR EACH LINE CAN BE FOUND BY CLICKING ON THE CELL LINE BELOW.

WHAT TO DO WHEN YOU RECEIVE YOUR CELL LINES

PYRAMID METHOD TO STOCK MAMMALIAN CELLS (upon receipt of your vial, this is the first thing you must do)

FETAL BOVINE SERUM INFORMATION - NOTE - FBS LOT WAS TESTED IN HCT116. IF YOU WILL BE USING THIS DISCOUNTED LOT (REQUIRED FOR GOLD STANDARD).

FOR ANY OTHER CELL LINES OR OTHER CONDITIONS (e.g. Dox-induction), PLEASE CONTACT TAKAYO SASAKI AT tsasaki@sdbri.org TO DISCUSS TESTING THE LOT WITH YOUR CONDITIONS.

ORDERING INFORMATION FOR FETAL BOVINE SERUM (FOR all Tier1 and Tier2 lines that use serum)

4DN PHASE2 FBS EVALUATION DATA

hPSC REAGENT INFORMATION

Stem Cell Tech Quote, which contains ordering instructions

Any new PIs joining 4DN recently who are not on this quote can be added to this quote. Please contact Takayo Sasaki tsasaki@sdbri.org

KARYOTYPE INFORMATION FOR NON-COMMERCIALLY STOCKED LINES (HFFc6, F121-9 and F123)

Document on how to read a karyotype report

HFFc6

F121-9

F123

FOR THE LATEST 4DN GROWTH SOP FOR EACH CELL LINE CLICK ON THE CELL LINE IN THE ABOVE LIST OF TIER1 AND TIER2 CELL LINES.

These protocols have been vetted by at least two 4DN laboratories and lots and prices for reagents negotiated. Following these SOPs is necessary for your data to be branded “gold standard”.

Differentiating hESCs to DE

Differentiating mESCs to NPCs

Differentiating mESCs to EpiLCs