title: GSM8966900: DES1_ATAC; Mus musculus; ATAC-seq
library_name: GSM8966900
library_construction_protocol: Aortic tissue was dissected from the aortic root to the brachiocephalic artery (Ascending), from the brachiocephalic artery up including the carotid artery (carotid), and descending thoracic aorta from subclavian artery to renal arter (descending). Protocol follows as such:1) Prepare the Enzyme Solution (on ice):     Final (working) concentration of Liberase is 2 units/mL    Final (working) concentration of Elastase is 2 units/mL     For 1mL of enzyme solution:     Liberase TM (26 units/mL)  77  uL     Elastase (125 units/mL)   16  uL     HBSS      907 uL     Total      1   mL  2) Place enzyme mixture on ice while aorta is being harvested  3) Perfuse mouse with ~10mL PBS via LV puncture  4) Harvest aortic root and open longitudinally. Wash with PBS x3 in 12-well dish. Keep on ice while collecting from multiple mice.  5) Move root to 12-well dish with 1mL of enzyme mixture.  6) Incubate in 37C incubator for ~30-45 minutes (adjust time based on age and extent of calcification e.g. if Apoe w/ 16wk HFD incubate for 45 minutes). At the end of the incubation, mince thoroughly with scissors for a few minutes (<5min) until tissue is mostly dissolved.  7) Pipet up and down ~20-30 times to thoroughly disrupt tissue. Confirm full digestion under the microscope (single cells w/o clumps). If the tissue is not fully digested after pipetting incubate at 37C for an additional 10-15 minutes (may lead to increased cell lysis).   8) Transfer cell suspension into 1.5mL Eppendorf and centrifuge at 500xg for 5 minutes.  9) Carefully remove supernatant (confirm that the cell pellet is still present).  10) Resuspend the cells in 200uL HBSS (adjust volume as needed). Cell solution was then sorted for live cells on FACS and then proceeded to 10X capture following 10X protocol.  11) Immediately prior to FACS and/or staining, pass cells through a 35um strainer. Note: Coating collection tubes in BSA solution may improve cell survival. 1) Coat the 1.5mL eppendorf tubes used to store the cells prior to FACS, and the FACS collection eppendorf tubes, with 10% BSA in PBS (i.e. fill up tube, and pipette out all volume - then wait a few minutes and then pipet out the rest that gradually slid to the bottom).  2) Wash Buffer: 10mM Tris pH 7.4 (CATALOGUE NUMBER) 10mM NaCl (CATALOGUE NUMBER) 3mM MgCl2 (CATALOGUE NUMBER) 1% BSA (1:10 dilution from 10% BSA stock (CATALOGUE NUMBER)) 0.1% Tween-20 (1:100 dilution from 10% stock (CATALOGUE NUMBER))  NOTE: Wash buffer can be prepared ahead of time and kept at 4C without adding BSA. Add all reagents, except for BSA, to a new 500mL bottle of ultra-pure water and keep at 4C until needed.  3) 0.1X lysis buffer: 196uL wash buffer + 2uL 1X Promega digitonin and 2uL NP-40, followed by 1:10 (200uL + 1800uL) dilution with wash buffer.   After FACS (all centrifugation steps at 4C):  4) Spin cells at 500g x 10 min, gently pipet off all supernatant (with P1000 and P100).  5) Add 45uL 0.1X lysis buffer, mix x3, and incubate for 15 minutes on ice.  6) Add 50uL chilled wash buffer WITHOUT mixing.  7) Spin at 500g x10 min, remove 95uL supernatant with P100.  8) Add 45uL 1X diluted nuclei buffer (diluted 1:20 with H2O), WITHOUT mixing.  9) Spin at 500g x10 min. Remove supernatant with P100 (set to 40uL) and P10 (set to 10uL).  10) Resuspend in 7uL 1X diluted nuclei buffer.  11) Mix 25uL nuclei buffer and 25uL Trypan blue. Mix 1 uL nuclei suspension with 9uL od Trypan blue dilution (1:10 dilution). Load into hemacytometer and examine with Zeiss in microscope room (not TC scope). Count the average number of nuclei in the large boxes and calculate nuclei/uL. Remember to account for 10x dilution factor.  12) Dilute nuclei with more nuclei buffer, if desired.  13) Aim for 8000-9000 nuclei per capture scRNA and scATAC libraries were prepared following 10X protocol.