Prior to the sorting, HeLa cells were trypsinized and resuspended in PBS with 5 mM EDTA. Then, cells were sorted on a SY3200 cell sorter (Sony Biotechnology)into 96-well plate which contained 3 μL of lysis Buffer. The plate was previously prepared with the lysis buffer and stored at -70°C. After the sorting, the plate was immediately frozen in carbonic ice and sent for RNA sequencing. Full-length single cell RNA sequencing libraries are prepared using the Smart-seq2 protocol (Picelli et al., Nat. Protoc. 2014) with minor modifications. Briefly, freshly harvested cells were sorted into 96-well plates containing the lysis buffer. Reverse transcription was performed using SuperScrpit II (Invitrogen) in the presence of oligo-dT30VN, template-switching oligonucleotides and betaine. The cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Kappa Biosystems), ISPCR primer and 23 cycles of amplification. Following purification with Agencourt Ampure XP beads (Beckmann Coulter), product size distribution and quantity are assessed on a Bioanalyzer using a High Sensitvity DNA Kit (Agilent Technologies). 150 pg of the amplified cDNA were fragmented using Nextera XT (Illumina) and amplified with indexed Nextera PCR primers. Products were purified twice with Agencourt Ampure XP beads and quantified again using a Bioanalyzer High Sensitvity DNA Kit. Sequencing of Nextera libraries was carried out on a Illumina HiSeq2500 v4.
GEO Accession
GSM3656238