GSM7105058: RNAseq_new_Aged1; Mus musculus; RNA-Seq
Fus low and Fus high HSCs(lineage-, c-kit+ Sca-1+ CD34-) from bone marrow of mice using fus gtp; Young(2 months) and Aged(30 months) HSCs(lineage-, c-kit+ Sca-1+ CD34-) from bone marrow of mice Hi-C: We performed in tagHi-C on FUSlow HSCs and FUShigh HSCs(Cell reports 32, 108206 (2020)). In briefly, 20,000 HSCs were sorted and crosslinked in 100 uL 1% formaldehyde for 10 minutes at room temperature. Cell pellet was collected by centrifuged for 5 minutes at 3,000 g at 4℃ and lysed in 25uL of Hi-C lysis buffer. Next MboI restriction enzyme in 2.5uL of 10x NEB buffer 2 were added to digest chromatin. Then DNA fragments were ligated by incubated in Ligation master mix (66.3 uL of ddH2O, 12 uL of 10 x T4 DNA ligase buffer, 10 uL of 10% (v/v) Triton X-100, 1.2 uL of 100 x BSA and 1 uL of 400 U/uL T4 DNA ligase).Next, DNA was extracted by150 uL Phenol-Chloroform-Isoamyl alcohol (25:24:1). Next, 20 uL of DNA fragmentation mix (4 uL of 5 x TTBL, 5 uL of TTE Mix V1, 5 ng DNA and ddH2O) was used for PCR amplification. After amplification, the samples were purified with AMPure beads once and resolved with ddH2O to prepare for sequencing library after the DNA concentration was quantified with Qubit. The libraries were sequenced using an Illumina Hiseq platform with 150-bp paired-end. CUT&RUN: Young HSCs and Aged HSCs were sorted from 2-month mice and 30-month mice. 100,000 HSCs were centrifuged at 1,000g for 5min at 4℃, and washed with 200uL PBS.le. Then the conA beads were separated by magnet and resuspended with 200 μl Antibody Buffer (2mM EDTA, 0.02% digitonin and CTCF antibody (1:250, EMD Millipore, 07-729) in washing buffer)》 Next, DNA was digested using 200 μL pA-MNase Buffer. Next, Endprep reaction system(25uL DNA and H2O, 1.5 μLNEB Next Ultra II Endprep Enzyme Mix, 3.5uLNEB Next Ultra II Endprep Reaction Buffer) was subjected to PCR amplification. After amplification, the samples were purified with 0.3× -0.6× VATHS Beads and resolved with ddH2O to prepare for sequencing library after the DNA concentration was quantified with Qubit. The libraries were sequenced using an Illumina Hiseq platform with 150-bp paired-end. CUT&Tag: FUSlow HSCs and FUShigh LSK cells were sorted from 2-4 month fus-gfp mice. 50,000 LSK cells were collected at 500g for 5min at RT, and performed according to the protocol of CUT&Tag Assay Kit(TD903, Vazyme) RNA-seq: First, 100 HSCs (Lineage- Sca-1+ c-kit+ CD34- GFPlow/high) from Fus-gfp mice were sorted into lysis buffer directly. The transcriptome libraries were prepared using Smart-seq2 method. Then, Agilent Bioanalyzer 2100 system (Agilent Technologies) was used to assess the insertion size, and HiSeq PE Cluster Kit v4-cBot-HS (Illumina) was used to perform the clustering of the index-coded samples according the manufacturer's instructions. Finally, the libraries were sequenced using an Illumina Hiseq platform with 150-bp paired-end.