title: GSM4115950: ESC_4i_Par_noIAA_r1; Homo sapiens; RNA-Seq
library_construction_protocol: RNA-seq was performed on PRDM14-AID-Venus competent 4i hESCs and hPGCLCs induced therefrom. Two biological replicates were used for each condition. For IAA-sensitive cells, two clones (cl11 and cl21) from the same hESC passage or the same hPGCLC induction were used as replicates. For the parental (“no TIR1”) control, the same cell line was used at different passages or inductions to yield two independent replicates. 10,000 AP+ 4i hESCs or 10,000 NANOS3-tdTomato+AP+ hPGCLCs (with the exception of hPGCLC cl21 replicate, where 3,000 cells were used) were sorted directly into 100 μl of extraction buffer from PicoPure RNA Isolation Kit (Applied Biosystems) for subsequent total RNA extraction according to manufacturer's protocol. RNA was stored at -80 ºC and its quality and quantity were checked by Agilent RNA 6000 Pico Kit with Bioanalyzer (Agilent Technologies) and Qubit (Thermo Fisher Scientific). RNA-seq library was prepared from 10 ng input RNA using the end-to-end Trio RNA-seq library prep kit (Nugen) following the manufacturer's protocol but omitting the AnyDeplete step. In short, the protocol contains the following steps: DNase treatment to remove DNA from RNA; first strand and second strand cDNA synthesis to produce the reverse complement of the input RNAs; cDNA purification using Agencourt AMPure XP beads (Backman Coulter); single primer isothermal amplification (SPIA) to stoichiometrically amplify cDNAs; enzymatic fragmentation and end repair; sequencing adaptor (index) ligation; product purification using AMPure beads; library amplification (4 cycles were used); library purification using AMPure beads. Libraries were then quantified by qPCR using NEBNExt Library Quant Kit (NEB) for Illumina on QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Fragment size distribution and the absence of adapter dimers was checked using Agilent TapeStation 2200 and High Sensitivity D1000 ScreenTape. Finally, RNA-seq libraries were subjected to single-end 50 bp sequencing on HiSeq 4000 sequencing system (Illumina). 24 indexed libraries (including samples from this work and 8 others) were multiplexed together and sequenced in two lanes of a flowcell, resulting in >30 million reads per sample.
attributes.tag: GEO Accession
attributes.value: GSM4115950