Library |
Library name |
Construction protocol |
Strategy |
Source |
Selection |
Layout |
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After extraction of total RNA from HRECs using RNeasy Mini kits (QIAGEN), the RNA purity was checked using a K5500 spectrophotometer (Kaiao, Beijing,China). RNA integrityand concentration were assessed using an RNA Nano 6000 Assay Kit of a Bioanalyzer 2100 system (Agilent Technologies,CA).Forlibrarypreparation,atotalof2 μgRNApersamplewas used as input material for the RNA sample preparations. Sequencing libraries were generated using NEB Next, Ultra RNA Library Prep Kit for Illumina® (Cat# E7530L, NEB, USA), following the 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 conditions in NEBNext First Strand Synthesis Reaction Buffer (5×). First-strand cDNA was synthesized using random hexamer primers and RNase H. Second-strand cDNA synthesis was subsequently performed using dNTPs,DNApolymeraseI andRNaseH.Thelibraryfragmentswerepurified with QiaQuick PCR kits and elution with EB buffer, and then terminal repair, A-tailing and addition of an adapter were implemented. The RNA concentration of the library was measured using a Qubit RNA Assay Kit in Qubit 3.0 to preliminarily quantify and then dilute to 1 ng/μl. Insert size was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies), and the qualified insert size was accurately quantified using a StepOnePlus™ Real-TimePCRSystem(libraryvalidconcentration>10 nM).Theclusteringof the index-coded samples was performed on a cBot cluster generation system using a HiSeq PE Cluster Kit v4-cBot-HS (Illumina, CA, USA) according to the manufacturer’s instructions. After cluster generation, the libraries were sequenced on an Illumina HiSeq 2500 platform, and 150 bp paired-end reads were generated. |
RNA-Seq |
TRANSCRIPTOMIC |
unspecified |
PAIRED
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