Kapa hyper pre kit

Single‐cell RNA‐seq libraries were prepared by using a modified protocol based on the STRT‐seq protocol. Briefly, after tissue digestion, single cells were picked into 2 μl cell lysis buffer with a mouth pipette under a microscope. Reverse transcription was performed with oligo dT primers composed of 8 nt cell‐specific barcodes, 8 nt unique molecular identifiers (UMI) and 25 nt oligo dT. The second‐strand cDNA was synthesized followed by 19 cycles of PCR amplification with the 3’P2 primer and the IS primer. Then, 96 different barcoded single‐cell PCR pre‐amplified products were pooled together and purified by AMPure XP beads (Beckman). Forty nanograms of DNA was then used to process four cycles PCR with IS primer and biotin‐modified index primer. Index‐induced cDNA was sheared to ~300 bp fragments by Covaris S2. Fragmented DNA was then enriched by incubating with streptavidin C1 beads (Thermo Fisher) for 1 h. Finally, the libraries were constructed using a KAPA Hyper Pre Kit (KAPA Biosystems). All single‐cell RNA‐seq data were generated on an Illumina HiSeq4000 platform for 150‐bp paired‐end reads.

Single-cell RNA-seq libraries were prepared using an experimental protocol based on single-cell transcriptomics sequencing technology. The in vivo samples were treated with acid Tyrode’s solution to remove zona pellucida. The morulae were transferred into 2 μL of lysate buffer under a microscope by pipetting. Reverse transcription was performed using PCR to synthesize second-strand cDNA. The 3′ P2 primers and IS primers were used to amplify the product with 16 PCR cycles. Subsequently, the 96 different barcode labeled single embryo PCR pre-amplification products were pooled and purified using AMPure XP magnetic beads. PCR amplification was then performed with 40 ng of DNA and IS primers. The amplified cDNA was broken into approximately 300 bp fragments using Covaris S2. DNA Fragments were then enriched by incubation with streptavidin C1 magnetic beads (Thermo Fisher) for 1 h. Finally, libraries were constructed using the KAPA Hyper Pre Kit (KAPA Biosystems). All single-cell RNA-seq data were generated on the Illumina HiSeq4000 platform for 150 bp paired-end reads.

Single-cell RNA-seq libraries were prepared using an experimental protocol based on single-cell transcriptomics sequencing technology. The in vivo samples were treated with acid Tyrode’s solution to remove zona pellucida. The morulae were transferred into 2 μL of lysate buffer under a microscope by pipetting. Reverse transcription was performed using PCR to synthesize second-strand cDNA. The 3′ P2 primers and IS primers were used to amplify the product with 16 PCR cycles. Subsequently, the 96 different barcode labeled single embryo PCR pre-amplification products were pooled and purified using AMPure XP magnetic beads. PCR amplification was then performed with 40 ng of DNA and IS primers. The amplified cDNA was broken into approximately 300 bp fragments using Covaris S2. DNA Fragments were then enriched by incubation with streptavidin C1 magnetic beads (Thermo Fisher) for 1 h. Finally, libraries were constructed using the KAPA Hyper Pre Kit (KAPA Biosystems). All single-cell RNA-seq data were generated on the Illumina HiSeq4000 platform for 150 bp paired-end reads.