GSM3956133
GSM3956133: C18; Homo sapiens; RNA-Seq
Total RNA extraction was performed using NucleoSpin RNA XS kit (Clontech Laboratories Inc. Cat. No. 740902). Briefly, cells were detached as mentioned in previous section, snap frozen in liquid nitrogen and stored at -80oC overnight. Next day, RNA was prepared from a total of 168 samples across three conditions (C, M and F) according to the manufacturer's instructions. cDNA library was prepared using SMART-Seq v4 3' DE kit according to the manufacturer's instructions (Clontech Laboratories Inc. Cat. No. 635040). For each clone, 1 ng of total RNA was used as input to synthesize cDNA which was subsequently amplified. Control RNA was provided with the kit. All samples were processed concurrently during library preparation. Prior to purification, the amplified cDNA's were grouped in fourteen pools of twelve samples each with a unique barcode (Clonetech Oligo dT in-line indexes or IL 1-12) ligated to the 3'end during cDNA synthesis. Pooled and purified libraries were fragmented using Nextera XT DNA Sample Preparation Kit (Illumina, Cat. Nos. FC-131-1024, FC-131- 1096). Illumina sequencing indexes (provided with the SMART-Seq v4 3' DE kit) were added to the fragmented libraries and indexed libraries were amplified. For intermittent purification steps, Agencourt AMPure XP PCR purification kit (5 ml Beckman Coulter Part No. A63880; 60 ml Beckman Coulter Part No. A63881) was used.
GEO
GSM3956133