| 生长方案: |
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| 处理方案: |
Informed consent was obtained from all patients preoperatively. Multi-regional sampling tissues were collected. For each tumor, 3-6 regions, including both surface and center areas, were sampled. Except for the MP of CRC01 which was sampled after six cycles of chemotherapy, other tumors were all sampled before treatment. Patient-derived tumors and adjacent normal tissue were collected and processed immediately after surgical resection. The dissected tissues were then mechanically dissociated and enzymatically digested to single-cell suspension using collagenase type II (Invitrogen, cat. #17101015) and collagenase type IV (Invitrogen, cat. #17104019). For 5 patients (CRC11, CRC12, CRC13, CRC14, and CRC15), leukocytes were depleted by magnetic-activated cell sorting (MACS) (CD45 Microbeads, Miltenyi Biotec, cat. #130-045-801) or fluorescence-activated cell sorting (FACS) (BV421 Mouse Anti-Human CD45, BD Horizon, cat. #563879). |
| 提取方案: |
The single viable cells were individually picked into 200-uL tubes containing lysis buffer. For scTrio-seq2, we used magnetic beads (Invitrogen, cat. #65011) to separate the nucleus and RNA of one single cell. We added 0.2 ?L magnetic beads to each single-cell lysis buffer. Then the single cells were lysed and vortexed for 1 min to release RNA. The lysis products were then centrifuged at 1,000 × g for 5 min at 4°C, and placed on the magnetic rack for 5 min. The magnetic beads can aggregate on the surface of the nucleus to maintain the nucleus in the pellet, while the RNA was released in the supernatants. The supernatants containing RNA were transferred to a new tube for transcriptome sequencing. The remaining beads containing a single nucleus were re-suspended with lysis buffer of scBS-seq for DNA methylation sequencing" |
| 建库方案: |
For single-cell whole-genome bisulfite sequencing, the scBS-seq libraries were constructed according to the published protocol as described in 2017 (Clark et al., 2017). For CRC01 and CRC02, the transcriptome sequencing libraries were constructed according to Tang protocol (Tang et al., 2009); for the remaining patients, the transcriptome sequencing libraries were constructed according to a multiplexed scRNA-seq method, in which the poly T primers were combined with barcodes and unique molecule identifiers (UMIs) (Dong et al., 2018)." |
