Illumina HiSeq 3000 sequencing; Specification of HiPS to in vivo placental cell types
Specification of HiPS to in vivo placental cell types
Barcoded beads (Chemgenes, # Macosko-2011-10) were washed with 30 mL of ethanol (Sigma Aldrich, # E7023-500ML), then removed all ethanol after centrifugation at 1,000g for 1 minute. Washed beads were suspended in 30 mL of TE-TW (10 mM Tris pH8.0, 1 mM EDTA, 0.5% SDS) and spun down at 1,000g for 1 minute. TE-TW was discarded and re-suspended in 20 mL of TE-TW. 100 µm strainer (Corning, Cat# 352360) was used for size selection of beads. Beads were suspended as the concentration of 120 beads/µL in 1 mL of the lysis buffer. To generate droplets, cells, beads, and oil (BioRad, #186-4006) were loaded into the 125-μm microfluidic device after aquapel flush treatment (FlowJEM). The cells and barcoded beads suspension were flown at 2 mL/h each and at 13 mL/h for oil.  Droplets were collected into the 50 mL tubes and 30 mL of 6X SSC was added, then broken with 1 mL of perfluorooctanol (Sigma, # 370533). Collected beads were washed with 1 mL of 6X SSC twice and with 300 µLof RT buffer. On removing the RT buffer, reverse transcription mixture was added into the beads. To synthesize cDNA, beads in RT mix were incubated with rotation at room temperature for 30 minutes and 42 °C for 90 minutes. The reverse transcription mixture was prepared as in the reference, Macosko, E.Z. et. al., Cell 161, 1202-1214 (2015). Beads were washed once with 1 mL of TE-SDS, twice with 1 mL of TE-TW, and with 1 mL of 10 mM Tris pH 8.0. Exonuclease I treatment was performed to remove excess bead primers at 37 °C for 45 minutes with rotation followed by washing beads once with 1 mL of TE-SDS, twice with 1 mL of TE-TW, and with 1 mL of molecular grade water. Unique barcode STAMPs (Single-cell Transcriptomes Attached to MicroParticles) were resuspended in 1 mL H2O, and loaded on a hemocytometer (Incyto, # DHC-N01). Aliquots of 3,000 beads in 24.6 µL of H2O were amplified with 0.8 µM SMART PCR primer (IDT, 5'-AAG CAG TGG TAT CAA CGC AGA GT-3') and 2X KAPA HiFi Hotstart Ready Mix (Fisher Scientific, # KK2602) using the following PCR condition: (step 1) 95 °C for 3 min, (step 2) four cycles of: 98 °C for 20 sec, 65 °C for 45 sec, 72 °C for 3 min, (step 3) 10 cycles of: 98 °C for 20 sec, 67 °C for 20 s, 72 °C for 3 min, and (step 4) finally, 72 °C for 5 min. Purified cDNA with 0.6X SPRI beads were analyzed with High Sensitive DNA kit (Agilent, # 5067-4626), then tagmented and amplified using Nextera XT Library Preparation Kit (Illumina, # FC-131-1096) by manufacturer's manual for sequencing.