Illumina NovaSeq 6000 paired end sequencing; Single cell analysis of MNX1 overexpression in hemogenic gastruloids
Single cell analysis of MNX1 overexpression in hemogenic gastruloids
At 216h, cells were stained with  C-Kit-APC-Cy7 (clone 2B8, Biolegend), CD41-PE (MWReg30, Biolegend), and CD45-APC (clone 30-F11, Biolegend), and deposited into 2 96-well plates as CD41+ and/or CD45+ cells. Cells were deposited into a lysis buffer as per the published Smart-Seq2 sequencing protocol, centrifuged and snap-frozen.  Cell lysis with 0.2% Triton X-100, 1U/µl RNase inhibitor, Thermo Fisher Scientific Reverse transcription was performed using SuperScript II (Thermo Fisher Scientific) in the presence of 1 μM oligo-dT30VN (IDT), 1 μM Template-switching oligonucleotides (QIAGEN), and 1 M betaine. cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Kapa Biosystems) and IS PCR primer (IDT), with 22 cycles of amplification. Following purification with Agencourt Ampure XP beads (Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer using a High Sensitivity DNA Kit (Agilent Technologies). A total of 140 pg of the amplified cDNA was fragmented using Nextera XT (Illumina) and amplified with with double indexed Nextera PCR primers (IDT). Products of each well of the 96-well plate were pooled and purified twice with Agencourt Ampure XP beads (Beckmann Coulter). Final libraries were quantified and checked for fragment size distribution using a Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies).