GSM8558860: RNA_IMR90_A_110; Homo sapiens; RNA-Seq
Single cells were collected into 0.2 ml PCR tubes by cell sorter or manually picked and lysed in RamDA-seq lysis buffer (TOYOBO, #RMD-101) containing 2 mg/ml Dynabeads™ MyOne™ Carboxylic Acid (Invitrogen, #65011). Genomic DNA was trapped on the beads, while RNA was released into the lysis buffer. The isolated DNA on magnetic beads was subjected to whole-genome amplification (WGA) and Next-Generation Sequencing (NGS) library preparation, as described in scRepli-seq (Takahashi et al., Nat Genet 2019). The DNA-seq (scRepli-seq) data was used to assess genome-wide DNA copy numbers, karyotype G1-phase cells, and analyze the replication state of S-phase cells. In parallel, the RNA isolated in the supernatant was subjected to scRamDA-seq (Hayashi et al., Nat Commun 2018) using the GenNext®RamDA-seq™ Single Cell Kit (TOYOBO, #RMD-101) according to the manufacturer's instructions with modifications. Sample and library preparation for scRR-seq-DNA (scRepli-seq): Genomic DNA was subjected to WGA with a SeqPlex kit (Sigma, SEQXE) in a 30-µl reaction volume, per the manufacturer's instructions. Amplified gDNA was purified and size-selected with Agencourt AMPure XP SPRI beads (1.7× reaction volume), and the SEQXE adapter sequence was removed by the primer removal enzyme Eco57I (Sigma, SEQXE). The gDNA was then purified with Agencourt AMPure XP SPRI beads (2.0× reaction volume) and eluted in 20 µl of 1/10× Elution Buffer (Qiagen). The DNA fragment size peak should be within 150–200 bp, which was confirmed by a capillary electrophoresis system, MultiNA (Shimadzu). We could easily distinguish single cells from zero or two cells by quantification of DNA with MultiNA, which provided reassurance that the gDNA was indeed derived from single cells. Then NGS libraries were constructed using the NGS LTP Library Preparation Kit (KAPA, KK8232) with a SeqCap adapter kit A/B (Roche, 07141530001/07141548001) or NEXTflex DNA barcode (Bio Scientific, NOVA), or the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England BioLabs , #E7645) with NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1, Set 2) (New England BioLabs , #E7600S, #E7780S ) or NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1, Set 2, Set 3, Set 4) (New England BioLabs , #E7355L, E7500L, E7710L, E7730L). Finally, the samples were subjected to NGS on an Illumina HiSeq X Ten system or Novaseq X Plus system (150-bp length, paired-end read).  Library preparation for scRR-seq-RNA scRamDA-seq NGS libraries were generated by using the Nextera® XT Library Prep Kit 24 Samples (illumina, #15032350, #15032352) with Nextera® XT Index Kit v2 Set A 96 indexes-samples (Illumina, #15052163) according to the manufacturer's instructions with modifications. Finally, the samples were subjected to NGS on an Illumina HiSeq X Ten system or Novaseq X Plus system (150-bp length, paired-end read).