title: GSM9034739: bone marrow transplantation mouse,wide-type control, vector 1 month; Mus musculus; RNA-Seq
library_name: GSM9034739
library_construction_protocol: RNA quantification and qualification ①RNA degradation and contamination was monitored on 1% agarose gels. ② RNA purity was checked using the NanoPhotometer®spectrophotometer (IMPLEN, CA, USA) . ③RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 1µg RNA per sample was used as input material for the RNAsample preparations. Sequencing libraries were generated using NEBNext® UltraTMRNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer'srecommendations and index codes were added to attribute sequences to each sample.Briefly, mRNA was purified from total RNA using poly-T oligo-attachedmagnetic beads. Fragmentation was carried out using divalent cations under elevatedtemperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strandcDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H ). Second strand cDNA synthesis was subsequentlyperformed using DNA Polymerase I and RNase H. Remaining overhangs wereconverted into blunt ends via exonuclease/polymerase activities. After adenylation of3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure wereligated to prepare for hybridization. In order to select cDNA fragments ofpreferentially 250~300 bp in length, the library fragments were purified with AMPureXP system (Beckman Coulter, Beverly, USA). Then 3µlUSER Enzyme (NEB, USA)was used with size-selected, adaptor-ligated cDNA at 37°C for 15 m in followed by 5min at 95°Cbefore PCR. Then PCR was performed with Phusion High -Fidelity DNApolymerase, Universal PCR primers and Index (X) Primer. At last, PCR productswere purified (AMPure XP system) and library quality was assessed on the AgilentBioanalyzer 2100 system.
identifiers.namespace: GEO
identifiers.id: GSM9034739_r1