GSM7438428
Bulk-RNA sequencing library is constructed based on Smart-seq2. SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara catalog # 634890) was used and experiments were performed according to manufacturer's instructions. Briefly, sorted HSCs were incubated in lysis buffer with RNase inhibitor on dry ice for 1 minutes, and then thawed at room temperature. First-Strand Buffer, SMART-Seq v4 Oligonucleotide, 3' SMART-Seq CDS primer II and RNase inhibitor were added into the lysed cells and incubated at 72°C for 3 min before being put on ice. Incubating was performed after adding SMARTScribe II Reverse Transcriptase as following: 42°C for 90 minutes then 70°C for 10 minutes. Without purification, SeqAmp PCR Buffer, PCR Primer II A and SeqAmp DNA Polymerase were added for pre-amplification using thermal cycler for 12 cycles following the manufacturer's instruction. The amplified cDNA was then purified by 1.8× volume of SPRIselect beads (Beckman Coulter catalog # B23318). Next, 200pg cDNA was used for library preparation with Nextera XT DNA Library Preparation Kit (Illumina catalog # FC-131-1024) according to manufacturer's instruction.