GSM4629169: placental HE cells; Mus musculus; RNA-Seq
Target cells were captured using antibody by fluorescence activated cell sorting (FACS). For 10X Genomics data, single-cell suspensions were loaded on a Chromium Single Cell Controller (10 × Genomics) to generate single-cell gel beads in emulsion (GEMs) by using Single Cell 30 Library and Gel Bead Kit V2 (10 × Genomics, 120237). RNAs barcoding were obtained through reverse transcription in individual GEMs. Barcoded cDNAs were pooled and cleaned up using beads (Invitrogen, 37002D). Single-cell RNA-sequencing (scRNA-seq) libraries were prepared using Single Cell 30 Library Gel Bead Kit V2 (10× Genomics, 120237) following the manufacture's introduction. Sequencing was performed on an Illumina HiSeq x Ten with pair end 150 bp (PE150). For smart-seq2 data, the linearized RNA molecules were releasein a thermocycler at 72 °C for 3 min at first. Then, transfer the reactions immediately on ice. Add 2.85 µl RT mixture into every reaction with component as SuperScript II reverse transcriptase, RNase Inhibitor, 5× Superscript II first-strand buffer, dithiothreitol (DTT), betaine, MgCl2, and template switch oligo (TSO) primer. Spin down the 5.4 µl samples in the thermocycler at 25 °C for 5 min, 42 °C for 60 min, 50 °C for 30 min, and then 70 °C for 10 min, 4 °C hold. Prepare 7.5 µl PCR mixture (6.25 μL 2× KAPA HiFi HotStart ReadyMix, ISPCR oligo, and 3′ Anchored oligo) to add in every tube reaction, and amplify samples for 19 cycles. Pool together samples with different barcodes (up to 96 barcodes) and purify DNA. Then prepare the second round of amplification with bio-tinylated index primer and ISPCR oligo for an additional five cycles. Subsequently, purify the biotinylated cDNAs further with 0.8× Ampure XP beads and elute cDNA in 50 µl EB. Next, shear the cDNAs into approximately 300-bp fragments with sonicator.Enrich the cDNA fragments  by Dynabeads® MyOneTM Streptavi- din C1. Libraries were prepared with KAPA Hyper Prep Kits. Use the NEB U-shaped adapter for ligation. Finally, amplify the adapter-ligated fragments using an Illumina QP2 primer and a short universal primer for 9 cycles.
GEO Accession
GSM4629169