License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: Jul 24, 2019
Last Modified: Feb 26, 2020
PROTOCOL integer ID: 26140
Keywords: Genomics, Epigenomics, Single Cell, Biotechnology
High-throughput single cell genomic assays resolve the heterogeneity of cell states in complex tissues, however, the spatial orientation within the network of interconnected cells is lost. As cell localization is a necessary dimension in understanding complex tissues and disease states, we present a tool for highly scalable spatially-resolved single cell profiling of chromatin state. We use high density multiregional sampling to perform single-cell combinatorial indexing on Microbiopsies Assigned to Positions for the Assay for Transposase Accessible Chromatin (sciMAP-ATAC) to produce single-cell data of equivalent quality to non-spatial single-cell ATAC-seq.
Nextera DNA Flex Library PrepIllumina, Inc.Catalog #20018705
QIAquick PCR Purification KitQiagenCatalog #28106
Uniquely Indexed TransposomesContributed by users
Sci- Barcoded PCR PrimersContributed by users
Pitstop 2Sigma AldrichCatalog #SML1169
Tween-20: working stock is 10% (100X). Aliquots are stored at 4C.
IGEPAL-630: Prepare10% (v/v) stock made with diH20, store at Room Temperature (RT).
DAPI: Resuspend to 5 mg/mL in diH20. Aliquot and store at -20C.
Pitstop2: Resuspend in 3mM in DMSO. Aliquot and store at -20C.
Supplies List:
96-well PCR plates (Eppendorf, 951020427)
35 um cell strainer (VWR, 21008-948)
High Sensitivity DNA Chip (Agilent, 5067-4627)
Instrument List:
Table top centrifuge cooled to 4C with rotors for spinning 1) 96-well plates, and 2) 15 mL falcon tubes at 600 rcf
Fluorescence Activated Cell Sorter (FACS), we use Sony SH800S
Thermomixer with 96 well plate adapter (55C incubations at 300 rpm), we use Eppendorf Themomixer C
Real-Time PCR instrument (Bio-Rad CFX Connect)
DNA fluorometer or spectrophotometer (Qubit Fluorometer 2.0 is used in this protocol)
Agilent Bioanalyzer
Sequencing: NextSeq 500 using custom chemistry protocol
Before start instructions
Cryopreserved tissue sections: Prepare prior to sp-sciATACseq protocol start. Refer to "Cryopreserved tissue sectioning" protocol
Uniquely indexed transposomes (8 uM): Prepare and load prior to sp-sciATACseq protocol start. Refer to "sci Transposase Loading" protocol.
Sp-sci barcoded PCR primers: Prepare prior to sp-sciATACseq protocol start. Refer to "sci Barcoded PCR Primer Preparation" protocol.
Prepare Nuclei Isolation Buffer
1
Construct 50mL Nuclei Isolation Buffer (NIB):
Final
Concentration
Stock
Concentration
Volume of Stock
10 mM Tris HCl, pH 7.5
1M
Tris-HCl, pH7.5
500 uL
10 mM NaCl
5M
NaCl
100 uL
3mM MgCl2
1M
MgCl2
150 uL
0.1 % Igepal
10%
Igepal
500 uL
0.1 % Tween
10%
Tween
500 uL
ddH20
to 50mL (add 48.25mL)
Note
OPTIONAL: To prevent protease degradation, we also add 2 tablets of Pierce Preotease Inhibitor Tablets, EDTA-Free to NIB following construction. We then vortex to fully dissolve tablets.
Note
NIB is stable at 4 °C for at least 1 month without noticable degradation in library quality or nuclei dissociation ability.
Store NIB on ice throughout nuclei dissociation and preparation of tagmentation plates.
Isolate nuclei
2
Nuclei from cryopreserved histological sections
If sample is sourced from microbiopsy of a cryopreserved histological section, dissociate cells using NIB incubation and trituration (described below).
Note
Note
Isolation of nuclei is dependent on the sample being used. And optimization should be performed. Below we list two example nuclei isolation protocols to act as general use for cell culture and primary tissue samples. Tissue should follow a dounce homogenization protocol, while liquid cell cutures can be pelleted and resuspended directly in NIB.
This protocol is optimized for brain tissue microbiopsies. Additional optimization may need to be performed for other tissues.
1. Prepare 96-well plate(s) for microbiopsy punches
Pipette 100 uL NIB into each well. Number of wells corresponds to number of punches to be collected.
Seal plate and store on ice until ready to collect microbiopsies
2. Prepare instruments & tissue for collecting microbiopsies.
Transfer cryopreserved tissue sections from -80C freezer on to dry ice in an insulated container
Load Palkovitz punch handle with selected diameter punch (options: 250 um, 500 um, 750 um, 1 mm, 1.25 mm)
Prechill Palkovitz punch by placing the punch in dry ice
3. Collect microbiopsies in a cryostat at -20C
Place tissue cryosection slide in cryostat and allow ~1 min to acclimate
Locate region of interest and collect punch
Deposit punch in well of 96-well plate by depressing punch plunger. (Ensure that punch enters well)
Repeat for each region to be resected. Place each new punch in new well
Reseal 96-well plate(s)
Note
Note: Keep a record of 1) slide number, 2) punch location, and 3) well ID for each punch.
Annotating image at cryostat works well.
4. Dissociate and wash microbiopsies
Shake plate on ice for 1 hour at 80 rpm
Using a multi-channel pipettor, triturate each well 30x.
Note
Note: Pipette gently in order to reduce bubbles and to prevent nuclei shearing
Spin down plate for 10 min at 500 rcf at 4C
Using a multi-channel pipettor, aspirate 90 uL of supernatant.
Note
Note: Pellet will not be visible. Be careful to not touch sides of bottom while drawing off supernatant.
5. Dilute microbiopsy nuclei to desired concentration
Note
Note: We find that for microbiopsy punches from 200 um thick tissue /250 um biopsy punch results in (thousand nuclei):
Using the same gates as first sort, sort X nuclei per well into prepared second plate with modified sort settings:
"Single cell" rather than "Normal"
This leads to a higher abort count (less efficient sorting) but is more precise in quantification
Keep sorted samples on ice to prevent transposases cross-reacting with other nuclei.
Example gating strategy using the Sony SH800 Flow sorter:
Spin down plate at 500 rcf for 00:03:00 min at 4 °C to ensure nuclei are properly suspended in solution.
Note
Volume added, even by sorting 100 nuclei is minimal in our hands and does not require concentration adjustments.
Transposase Denaturation
8
Transposase Denaturation
Denature remaining transposase in sorted nuclei using SDS mixture on Eppendorf Thermocyclers.
55 °C for 00:20:00 min
96-plex PCR
9
Amplifying single cell libraries
Note
Nextera PCR Mater Mix currently produces the highest quality libraries. An alternative master mix using Kapa Hifi Non-Hotstart has been developed and produces good results.
Step 9 includes a Step case.
Using Nextera PCR Master Mix
Using Kapa Hifi Non-Hotstart
step case
Using Nextera PCR Master Mix
10
Add 13.5 uL PCR Master Mix to each well
7.5 µL NPM
0.25 µL 100X SYBR Green I
5.5 µL dH2O
Perform Real-time PCR on the Bio-Rad CFX Connect:
PCR protocol for Kapa Hifi Non-Hotstart Library Amplification
Pull once majority of well begin to plateau. Sci-ATAC libraries amplify between 14-22 cycles dependent on nuclei per well.
Store libraries at 4 °C for 6 months or -20 °C forever
Library Clean-up and Quantification
11
Pool post-PCR Product
Pool 10 uL from each well into 15mL conical tube.
12
Concentrate DNA via column clean up
Run full pool volume through Qiaquick PCR purification column following manufacturer's protocol.
Elute in 50 µL 10 mM Tris-HCl pH 8.0
13
Clean by size selection with SPRI beads
Perform a 1X SPRI bead size selection (selecting for DNA > 200 bp).
Add 50 µL 18% PEG SPRI Beads to column elution, once beads are at room temperature.
Let mixture incubate at room temperature for 00:05:00 min
Place tube on magnetic rack and wait for magnetic beads to pellet and elution to fully clear (roughly 00:02:00 min)
Remove full volume of elution without disrupting bead pellet.