GSM5453846: J-LatA2+SAHA (P2449_N724-S510); Homo sapiens; RNA-Seq
Single cells were sorted by FACSAria II (Beckman Courter, CA, USA) to 96-well plates filled with lysis buffer (0.2% Triton X-100, 1 U/ul RNase inhibitors). Reverse transcription was performed using SuperScript II (ThermoFisher Scientific) in the presence of 1 M betaine, 1 uM poly(T) primer with sequence 5'-T(30)VN-3', 1 uM template-switching oligonucleotide with sequence 5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3', where "rG" stands for a G with ribose instead of deoxyiribose, and "+" stands for a LNA bond (Locked Nucleic Acid). The thermo-cycling conditions were // 90 min 42C // 2 min 50C / 2 min 42C / x10 // 15 min 70C. cDNAs were amplified using the KAPA Hifi Hotstart ReadyMix (Kapa Biosystems) and IS PCR primer with sequence 5'-AAGCAGTGGTATCAACGCAGAGT-3' (IDT), with thermo-cycling conditions // 3 min 98C // 15 s 98C / 20 s 67C / 6 min 72C / x25 // 5 min 72C. 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). 140 pg of the amplified cDNA were fragmented using Nextera XT (Illumina) and amplified with Nextera XT indexes (Illumina). 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).