title: GSM3897718: Single-cell Hi-C data of shoot cell 3; Oryza sativa Japonica Group; Hi-C
library_construction_protocol: After isolation, cells were immediately aspirated with a glass micropipette and blown out to 0.53 M mannitol (pH 5.7, adjusted with 0.1 M MES) containing 2% (v/v) formaldehyde by using a glass micropipette system. After 15 minutes, the cells were aspirated and blown out to fresh 0.53 M mannitol without formaldehyde for washing, Finally, the fixed clean cells were aspirated one by one, and blown into a 200 μl PCR well containing 7.7 μl Dpn II buffer (1X), at 4°C and kept on ice for next steps. For the precise protocol for library construction, please see the paper of this data linked to. Briefly,7.7 μl 1×Dpn II buffer containing single cell was heated in 62 °C for 7 min to directly lyse cell and to loosen the nuclear membrane.For each well, 0.3 μl (3 U) Dpn II (R0543, NEB) was added after cooling, and incubate at 37 °C for 14 hours.Denature Dpn II at 62 °C for 10 min. Add 2 μl ligation mixture (1 μl 10×T4 ligase buffer, 1 μl (400 U) T4 DNA ligase, M0202, NEB),hold at 16 °C for 4.5 hours, then at room temperature for 30 min.Denature ligase and de-crosslink at 65 °C for 6 hours. DNA amplification was applied with REPLI-g Single Cell Kit (150345, QIAGEN).Shear DNA to 150-500 bp range by sonication.Finally,contruct the library for illumina sequencing by following the classic steps including end repairing, adaptor ligation, and DNA amplification.
attributes.tag: GEO Accession
attributes.value: GSM3897718