Bone marrow was harvested from the femur, tibia and pelvic bones of 8–10 week old, mixed sex C57BL/6 mice, pooled, and subjected to ammonium chloride mediated red blood cell lysis. The sample was stained with a Lineage/Sca1/Kit staining cocktail, and 10,000 Lin-Sca1+Kit- cells were FACS sorted into an Eppendorf tube, and DNA was extracted using AllPrep DNA/RNA Mini Kit (Qiagen, 80204) according to manufacturer’s instructions. 4 uL of purified DNA (25 ng/uL) was mixed with 1 uL of T7 Phage Lambda DNA spikeins (1 ng/uL), and the mixture was used directly as input to bisulfite conversion and desulphonation as described for single-cells, except neither DNase nor UV decontamination was performed on the reagents, and 5 uL of beads was used instead of 1.8 uL. After bisulfite conversion and desulphonation, the DNA was eluted in 40 uL of EB buffer before used as input for one round of double-stranding with 2.5 uL of 10 mM dNTPs, 2 uL of 500 uM random hexamers, 1 uL of 50 U/uL Klenow exo- (NEB, M0212M) and 5 uL of 10X NEB Buffer 2. Library construction was the same for single-cells except only 4 rounds of PCR was used. Libraries were ran on agilent and sequenced directly without qPCR quality control.
GEO Accession
GSM2522282