GSM3602373: tri-vol-d6-181118E; Homo sapiens; RNA-Seq
After pick single cells using mouth pipette, they were transferred to cold scRNA-seq lysis buffer immediately. Subsequently, the lysis buffer were shocked for 3min at 72℃ to make sure the release of RNA. We use a modified STRT protocol to construct our library. In brief, we use single cell-specific barcodes to insure the protocol was applicable to a large number of cells. After cDNA amplification, the single cells labeled with different barcodes as many as 48-96 cells can be pooled together for subsequent library construction and sequencing. What's more, we add biotin in our indexed primers which can pair with 3' cDNA primers, so after additional amplifying of pooled cDNAs following shearing to about 300bp , we use Dynabeads® MyOne™ Streptavidin C1 beads to enrich for the 3'. The RNA-seq library was constructed using a Kapa Hyper Prep Kit (Kapa Biosystems) and subjected to 150 bp paired-end sequencing on an Illumina HiSeq 4000 platform (sequenced by Novogene). Briefly, the single cells dissociated from the individual embryo were picked by mouth pipette and put into the lysis buffer. The procedure of cell lysis includes the following two steps: 50 °C for 3 hr of incubation, and 70 °C for 15 min to inactivate the protease. Full-length RNA sequencing libraries were constructed based on the modified STRT-seq with changes below. Products obtained after transcription and the cDNA amplification were directly proceeded to fragmentation by the Covaris S2 system. After purification, the libraries were eventually generated by the Kapa Hyper Prep kit (Kapa Biosystems), which were sequenced on the Illumina HiSeq 4000 platform.
GEO Accession
GSM3602373