GSM3373937: ICM3; Homo sapiens; RNA-Seq
The samples were processed directly using the SMARTer Ultra Low Input RNA for Illumina sequencing kit (Clontech Laboratories, Mountain View, CA, USA) to ensure minimum loss of cells. According to the manufacturer's suggested protocol, samples were collected in 2µl of DMEM-F12 + Glutamax (Invitrogen) with 25% bovine serum albumin (Sigma) and transferred to 5µl of pre-prepared reaction buffer. A final volume of 9µl was set up using nuclease-free water. The sample reaction mixture was then mixed with the first primer (3' SMART CDS primer II A) for first strand synthesis. This reaction mixture was placed in a thermocycler for 3 minutes and then incubated on ice. From this step, the first strand cDNA synthesis was set up directly using the SMARTScribe Reverse Transcriptase. Once completed, the first-strand cDNA was purified using SPRI Ampure beads, followed by double stranded (ds) cDNA amplification by long distance PCR. The amplified ds cDNA was then further purified using the Ampure beads. cDNA was fragmented in the 200-500bp range on a Covaris S2 Focused-ultrasonicator (Brighton, UK), according to the SMARTer Ultra Low Input RNA for Illumina sequencing protocol. A sequencing library was made using the Low Input Library Prep Kit (Clontech Laboratories, Mountain View, CA, USA).
GEO Accession
GSM3373937