design: After extraction of total RNA from HRECs using RNeasy Mini kits (Qiagen), the RNA purity was assessed using a K5500 spectrophotometer (Kaiao, Beijing, China). RNA integrity and concentration were assessed using an RNA Nano 6000 Assay Kit of a Bioanalyzer 2100 system (Agilent Technologies, CA, USA). For library preparation, a total of 2 g RNA per sample was 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 manufacturers 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 primers and RNase H. Second-strand cDNA synthesis was subsequently performed using dNTPs, DNA polymerase I and RNase H. The library fragments were purified with QiaQuick PCR kits and eluted 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 the sample and then dilute it to 1 ng/l. Insert size was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA), and the qualified insert size was accurately quantified using a StepOnePlus Real-Time PCR System (valid library concentration>10 nM). Clustering of 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 manufacturers instructions. After cluster generation, the libraries were sequenced on an Illumina HiSeq 2500 platform, and 150 bp paired-end reads were generated.