Total RNA was extracted from the iPRE maintained in KSR or B27 medium for 4 weeks via TRIzol™ Reagent (Invitrogen) mRNA was purified from total RNA via poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer (5X). First-strand cDNA was synthesized via random hexamer primers and M-MulV reverse transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed via DNA polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of the DNA fragments, adaptors with hairpin loop structures were ligated to prepare for hybridization. To preferentially select cDNA fragments 370-420 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) Primer. Then, the PCR products were purified, and library quality was assessed on an Agilent Bioanalyzer 2100 system. Clustering of the index-coded samples was performed on a cBot Cluster Generation System via the TruSeq PE Cluster Kit v3 - cBot - HS (Illumina). After cluster generation, the library preparations were sequenced on an Illumina NovaSeq platform, and 150 bp paired-end reads were generated.