GSM9072118: D1408T118; Homo sapiens; RNA-Seq
SMART-seq3: Single cell distribution of individual cell families has been performed using the cellenONE platform (Cellenion). Briefly, we selected individual wells of 96-wells plates displaying at least four cells, harvested the entire well, centrifugated for 8' at 300g and resuspended in 6 µL of PBS 1X (Sigma). Then, the whole volume was loaded into the machine, but due to the dead volume, effectively only 4 µL of this cell suspension could be redistributed. Individual cells were each sorted directly into single wells of 96-wells plates (90 cells per plate) at 4 °C, each well containing 3 µL of lysis buffer. The lysis buffer contains 0.03 µL Triton X100 (ThermoFisher Scientific), 0.0375 µL Recombinant RNAse Inhibitor (Takara Bio), 0.2 µL dNTP 10mM (ThermoFisher Scientific), 0.5 µL PEG 40% (Sigma), 0.04 µL SMARTseq3 oligo-dT 100 μM (/5BiosG/ACGAGCATCAGCAGCATACGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN; IDT) and ultrapure RNAse water (Invitrogen).  CITE-seq: Magnetic sorted CD34+ cells from umbilical cord blood were plated at a cell density of 1x105 cells/mL, using the same culture condition described for the condition Diff. Cells were harvested after 96 hours, counted with a haemocytometer, and resuspended in cold PBS 1X (ThermoFisher Scientific) containing 2% Bovine Serum Albumin (BSA, Euromedex) and 0.01% Tween 20 (Sigma), to a final concentration of 106 cells/mL. 100 µL of this cell suspension was incubated with a mix of previously prepared CITE-seq antibodies. Cells were incubated for 30 minutes at 4 °C, washed three times with the same solution used for the incubation and resuspended in 16.5 µL, at a final concentration of 1000 cells/mL. SMART-seq3: Reverse transcription (RT) and the subsequent steps were performed accordingly to the SMART-seq3 technique40. Briefly, lysates in each well underwent RT (42 °C for 90 mins, 10 cycles at 50 °C 2 mins + 42 °C 2 mins, and finally 85 °C for 5 mins) on ice, in presence of template switch oligonucleotide at 100 µM (/5BiosG/AGAGACAGATTGCGCAATGNNNNNNNNrGrGrGIDT), 0.04 µL Maxima H minus reverse transcriptase (ThermoFisher Scientific), TRIS-HCl 1M PH 8.3 (Invitrogen), 0.12µL NaCl 1M (Invitrogen), 0.04 µL GTP 100mM (ThermoFisher Scientific), 0,32 µL DTT 100mM (ThermoFisher Scientific), 0.1 µL MgCl2 50mM (ThermoFisher Scientific) and 0.05 µL Recombinant RNAse Inhibitor (Takara Bio) and water. The following PCR step was performed immediately after, using the Kapa Hifi HotStart Ready Mix (Roche), and forward (100 µM TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATTGCGCAATG) and reverse (25 µM ACGAGCATCAGCAGCATACGA) primers (IDT technologies) for 25 cycles. After DNA purification using AMPure XP microbeads (ratio 0.8:1) (Beckman Coulter, A63881), the cDNA was tagmented in a final volume of 2 µL, using the Nextera XT DNA Library preparation kit (Illumina). The following library amplification was performed adding 1.5 µL of UDI primers and 2,5 µL of PCR mix including the Phusion High Fidelity DNA polymerase (ThermoFisher Scientific). After tagmentation, the different samples were pooled, purified using AMPure XP microbeads (ratio 0.9:1) and sequenced at 100-bp paired end on a high-output flow cell, using a NovaSeq SP, 800M reads (Illumina).  CITE-seq: Approximately 1.65x104 cells were loaded, to obtain a final yield of 1x104 recovered cells. Cells were processed accordingly to the Chromium Single Cell 3' library v3.1 (10x Genomics). Libraries were sequenced using a Novaseq s1 (Illumina), allocating at least 20,000 reads per cell for the mRNA library, and 5,000 reads for the Antibody Derived Tags (ADT) library