organism: Homo sapiens
description: Protocols: Cells were harvested at day 2 of culture and sorted into a 15ml falcon tube at 500,000 cells per sample. Post sort, cells were centrifuged at 270 x g for 3 minutes, supernatant removed and cell pellets were stored at -80C prior to RNA extraction. Co-culture Total RNA was extracted using Qiagen RNAeasy Kit as per the manufacturers instructions. Total RNA was quality controlled and quantified using Qubit 3.0 (LifeTechnologies) and Nanodrop LITE spectrophotometer (ThermoFisher). mRNA from eukaryotic organisms was enriched using oligo(dT) beads and then fragmented randomly in fragmentation buffer, followed by cDNA A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer（5X. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase（RNase H- . Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter,Beverly, USA). Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: developmental stage
attributes.value: blastocyst
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: cell_type
attributes.value: pluripotent stem cell
attributes.tag: cell_line
attributes.value: H7v1,12,17q,20q-GFP
attributes.tag: karyotype
attributes.value: 48,XX,+del(1)(p22p22),der(6)t(6;17)(q27;q1),+12,ish dup(20)(q11.21q11.21)
attributes.tag: phenotype
attributes.value: competitive growth winner
attributes.tag: growth condition
attributes.value: co-culture
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-03-23
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2021-08-01