Reprogramming pZscan4-Emerald ESC derived XEN-like cells and FACS sorted pZscan4c-eme+ cells at stage II day 12
cell type
Intermediate cells
strain
129S6/SvEvTac x C57BL/6Ncr)
time
stage II day 12
Sequenced DNA Library
library_strategy
Bisulfite-Seq
library_source
GENOMIC
library_selection
RANDOM
library_construction_protocol
Total RNA was isolated using Direct-zol RNA MiniPrep Kit (Zymo Research). Genomic DNA of sorted pZscan4c-Em+, pZscan4c-Em- cells at stage II D12, XEN-like cell with pZscan4c-Emerald reporter and CiPSCs were extracted using EasyPure Genomic DNA Kit (TransGen, EE101-11). Total RNA was isolated using Direct-zol RNA MiniPrep Kit (Zymo Research). RNA sequencing libraries were constructed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB England BioLabs).. Genomic DNA of sorted pZscan4c-Em+, pZscan4c-Em- cells at stage II D12 and pZscan4c-Emerald XEN-like cell were extracted using EasyPure Genomic DNA Kit (TransGen, EE101-11). DNA were denatured and bisulfite converted using EZ DNA Methylation-GoldTM Kit (Zymo Research, D5005). In brief, 20 µl DNA (300 ng) was mixed with 130 µl CT Conversion Reagent and performming the following steps: 98°C for 10 minutes, 64°C for 2.5 hours. Samples and 600 µl M-Binding Buffer were added into a Zymo-Spin™ IC Column, followed by inverting the column several times and centrifuging at full speed (>10,000 x g) for 30 seconds. 100 µl M-Wash Buffer was added to the column and centrifuged at full speed for 30 seconds. Add 200 µl M-Desulphonation Buffer to the column, incubate at room temperature (20-30°C) for 15-20 minutes and then centrifuging at full speed for 30 seconds. Wash the column with 200 µl M-Wash Buffer twice and elute the DNA with 10 µl M-Elution Buffer. Recovered bisulfite-converted DNAs were constructed into sequencing libraries using Accel-NGS Methyl-Seq DNA Library Kit (Swift, 30024) following the manufacturer's instructions. Cells at different timepoints throughout chemical reprogramming process were harvested and resuspended at 1 × 106 cells per milliliter in 1 × PBS with 0.04% BSA. Then, cell suspensions (300-1000 living cells per microliter determined by trypan blue staining) were loaded on a Chromium Single Cell Controller (10x Genomics) to generate single-cell gel beads in emulsion (GEMs) by using Single Cell 3' Library and Gel Bead Kit V2 (10x Genomics, 120237). Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs (Zheng et al., 2017). Barcoded cDNAs were pooled and cleanup by using DynaBeads® MyOne™ Silane Beads (Invitrogen, 37002D). Single-cell RNA-seq libraries were prepared using Single Cell 3' Library Gel Bead Kit V2 (10x Genomics, 120237) following the manufacture's introduction.