title: Illumina NovaSeq 6000 single sequencing of SAMD00286406
library_construction_protocol: Cell enrichment was performed as follow. One ml of cancer cell-spiked PBS-EB (or blood) or patient blood was introduced into the PBS-EB pre-filled CTC recovery device equipped the microcavity array (PMID: 27299849). Subsequently, negative pressure was applied to the samples with a peristaltic pump (MINIPULS 3: Gilson, Wisconsin, USA) which was connected to a vacuum line. The sample was passed through the MCA at a flow rate of 150 ul per min. To remove any residual cells, 2 mL of PBS-EB was passed through the MCA at a flow rate of 200 uL per min for 10 min. For the patient blood samples, 1 mL PBS-EB containing 5 uM CellTracker Green CMFDA and 1 ug per ml Hoechst 33342 was introduced into the MCA and incubated for 20 min and the excess dye was washed with 1 mL PBS-EB. Then, RNA of recovered cells was stabilized with CellCover throughout the MCA for 15 min. After washing with 1 mL PBS-EB, cancer cell-spiked PBS-EB samples were moved to the GCM step. For patient blood samples, cells were stained with anti-CD45 antibody (Hitachi Chemical, Tokyo, Japan) and Texas Red-labeled secondary antibody (Hitachi Chemical). Finally, the array was washed with 1 mL of PBS-EB and PBS to remove excess antibodies. Single cell isolation was performed as follow. Single cells were isolated by gel-based cell manipulation (GCM). Briefly, prior to GCM, polyethylene glycol diacrylate (PEGDA, Mn = 700: Sigma-Aldrich) with 0.5 percent Irgacure 2959 (photoinitiator) was introduced onto the MCA. Then, the CTC recovery device was covered with a coverslip (24 x 40 mm: Matsunami Glass). Coverslips were modified with 3-(trimethoxysilyl)-propyl methacrylate to provide a stiff connection between the coverslip and hydrogel. The MCA was observed under a fluorescence microscope (BX53: Olympus Co.), and cancer cells and CTC-candidates were determined as CellTracker Green- and Hoechst-33342 positive and CD45-negative cells. The subsequent steps were performed as described previously (PMID: 27299849). Single-cells were encapsulated into the hydrogel by light irradiation (wavelength = 365 nm, 12.7 mW per cm2 for 30 s). After peeling off the coverslip, hydrogel-encapsulated single-cells were resuspended into 0.4 ul of lysis buffer (0.5 percent NP40) in 200 uL PCR tubes (N8010840, Thermo Fisher Scientific) using tweezers. Isolated single-cells were immediately frozen with liquid nitrogen and stored at -80C for further whole transcriptome amplification (WTA). Single-cell WTA was performed by the Quartz-seq protocol (PMID: 23594475). In brief, mRNA from single cells were captured by oligo dT linked to an adaptor sequence, and first-strand cDNA was synthesized by a reverse transcription reaction. After a primer digestion reaction for removal of excess primers, a poly-A-tailing reaction by terminal transferase on the 3-prime end of the cDNA and mRNA degradation reaction by RNaseH were performed. Then second-strand cDNA was synthesized using a oligo dT linked to an adaptor sequence and amplified by PCR (21 cycles) using primers that hybridize to the adaptor sequence. Amplified cDNA was purified by MinElute PCR Purification Kit (QIAGEN, Hilden, Germany) and eluted in 24 ul of buffer EB. Then, yield and average length of amplified cDNA were measured using High Sensitivity DNA Kit (Agilent, California, USA). For quality checks, amplification of GAPDH by qPCR using 50 pg of amplified cDNA as template was performed. Only cells with detectable levels of GAPDH were subjected to further qPCR and selected for library construction for RNA-seq analysis.Ten nano grams of amplified cDNA was used for library construction with NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs, Massachusetts, USA). The cDNA was sheared by the Covaris system, and then the sheared fragments were end-repaired, A-tailed, and ligated to sequencing adaptors according to manufacturers protocol. A size selection of about 200 bp was performed before the PCR enrichment (12 cycles). Concentration of the cDNA library was quantified using a Qubit 2.0 fluorometer (Thermo Fisher), and diluted to 2 ng per ul before checking the insert size on an Agilent 2100 and quantifying by qPCR for greater accuracy (library activity >2 nM). Libraries were subjected to Illumina Hiseq 2500 or Illumina Novaseq 6000 and 50-base single-ended reads to a depth of 5M reads per samples were obtained.