Single Cell Sequencing and Multidimensional Omics Studies in Cardiovascular and Neurological Diseases

Test population Disease-specific experimental cohort of cardiovascular and neurological diseases (atrial fibrillation secondary to valvular heart disease, carotid atherosclerosis, moyamoya disease) and corresponding control cohort of normal tissue.

Sample size calculation Through literature research and clinical diagnosis experience, reliable conclusions can be obtained with an experimental cohort of 200 or more people and a control cohort. So the experimental data were selected from a disease cohort of 200 people and a control cohort of 200 people.

Specific research content

Clinical data collection Clinical data (name, age, sex, weight/height ratio (BMI), three routine procedures (blood routine, stool routine, urine routine), blood biochemistry, liver and kidney function, coagulation function, bedside electrocardiogram, 24-hour holter electrocardiogram, heart color Doppler ultrasound, carotid color Doppler ultrasound, family history, past history, medication history, treatment history, and personal history) were collected from patients who met the inclusion criteria and controls Etc.), sign a written informed consent.

1. Blood collection: Collect 5-10ml of fasting blood from the experimental population in the morning for blood routine, blood biochemistry, liver and kidney function, coagulation function and other tests.

2. Fecal collection: 3-5g feces of the experimental population without eating from 6 a.m. to 9 a.m. will be collected for stool routine and intestinal flora detection. Note: The sample must not take antibiotics and other drugs within 45 days.

3. Urine collection: Collect the first urine of the experimental group in the morning, and reserve 10-12ml of fresh midstream urine for routine urine detection.

   After testing the samples collected above, the test results were put into the corresponding experimental population data.

Single cell sequencing

1. Sample collection and processing ① Sample collection:

   Sample collection of patients with atrial fibrillation:

   A small amount of useless left atrial appendage tissue 100mg was taken as a sample during surgical treatment of atrial fibrillation patients. (The tissue used in this study was either residual tissue or abandoned tissue, and both were necessary for surgical resection, so there was no additional risk to the subjects)

   Atrial fibrillation control heart tissue sample collection:

   Atrial fibrillation control heart tissue was 100mg of cut waste tissue from heart transplant donor heart.

   Carotid atherosclerosis sample collection:

   During the surgical treatment of carotid atherosclerotic plaque endarterectomy, at least 1mg of the central tissue of discarded vascular plaque was collected as a sample.

   Carotid atherosclerosis control sample collection:

   During the surgical treatment of carotid atherosclerotic plaque endarterectomy, at least 1mg of marginal tissue of discarded vascular plaque was collected as control samples.

   Moyamoya disease sample collection:

   During cerebrovascular bypass surgery for Moyamoya disease, at least 1mg of discarded vascular tissue was collected as a sample.

   Moyamoya disease control sample collection:

   During cerebrovascular bypass surgery in non-moyamoya disease patients, at least 1mg of pruned waste vascular tissue was collected as a sample.

   • Sample preservation and delivery: Considering that on-site experiments need to be booked in advance and coordinated, and there are many uncertain factors in clinical sample collection. For most freshly obtained samples, Miltenyi MACS Tissue Storage Solution® (130-100-008) is recommended. Optimized for the storage of fresh organ and tissue samples, the preservation solution has been tested and validated on a variety of human and mouse tissues, including tumors, skin, heart, spleen, brain, and skeletal muscle.

   Procedure for using the preservation solution: a) Collect fresh samples from the operating room or anatomy, and quickly remove non-target tissues such as fat, hair, fibers, necrotic parts, etc., within 10 minutes; b) If the tissue block is large, it is recommended to divide it into soybean size and save it or keep it for backup; c) Directly transfer the sample to the pre-cooled (2-8℃) tissue preservation solution (1.5ml EP tube) to ensure that the tissue blocks are completely covered and the sample name is well marked; d) Seal the sample tube with sealing film, separate the sample from the ice pack with foam paper, put it in a ziplock bag, and arrange the ice pack to be sent as soon as possible. a) The tissue storage solution can be stored for 48h. In order to ensure the quality of the sample, the logistics delivery should be delivered the next day or the next morning, and the mailing should be delivered in advance of the sample date. b) It is recommended to place enough ice packs to avoid complete melting, and to avoid direct contact with the sample tube, causing the sample to freeze; c) Select a foam box with a thickness of not less than 3cm, and put some ice packs into the bottom first; d) The sample tube and the ice pack should be separated by foam paper or bubble bag; It is necessary to prepare the ice pack in advance, and avoid taking the ice bag out directly from -80℃ and -20℃, otherwise the tissue block will freeze due to low temperature, resulting in tissue damage.

   ③ Digestion: Fresh heart tissue (no less than 100mg of control clipped tissue, 3-4 pieces of puncture tissue (16G puncture needle, 1.5cm)), chopped into 1~3mm3 tissue blocks, And transferred to 15 ml EP tubes of DMEM containing collagenase I (450 U ML-1), DNase I (60 U ML-1) and hyaluronidase (60 U ML-1). Incubate at 37 ° C for 1 hour, then add HBB buffer (2% FBS and 0.2% BSA in HBSS) to stop digestion, filter through a 40 µm filter into a 50 ml conical tube, transfer to a clean 15 ml EP tube and centrifuge at 4 ° C 350g for 5 min. The supernatant was then removed and the particles were re-suspended in 1 ml of ACK lysis buffer (Gibco, A10492), digested at room temperature for 5 minutes before 9 ml of DMEM was added. The supernatant was removed by centrifugation and re-suspended in 5 ml FACS buffer (2% FBS and 2mM EDTA, in a calcium/magnesium free PBS).

   ④ Sorting: Centrifugation was repeated under the above conditions, the supernatant was removed, and the cell particles were re-suspended in 300 µl cell suspension buffer (0.04%BSA, 1×PBS) and 1 µl DRAQ5 (Thermo Fisher Scientific, 62251) and 4, 6-diaminidine - 2-phenylindole (DAPI; BD Biosciences, 564907) and incubate for 5 minutes before sorting. DRAQ5+/DAPI- cells are collected in a cell re-suspension buffer. The collected cells were then re-centrifuged according to the above parameters and re-suspended in a cell re-suspension buffer to a target concentration of 1,000 cells ul-1. The cells are counted with a blood cell counter and the concentration is adjusted as needed.

   ⑤ Database building: All reagents required for database building are included in 10X Genomics Single Cell 3 'Reagent Kits v2 kit or BD database building kit.