title: Illumina HiSeq 2500 sequencing; SLIC-CAGE (Super-Low Input Carrier-CAGE) transcription start site mapping of mouse migratory and gonadal primordial germ cells, postnatal oocytes and early embryo
design: SLIC-CAGE (Super-Low Input Carrier-CAGE) transcription start site mapping of mouse migratory and gonadal primordial germ cells, postnatal oocytes and early embryo
library_construction_protocol: Primordial and gonadal germ cells were isolated from embryos obtained from a 129Sv female and GOF18ΔPE-EGFP male cross. Genital ridges from 1-2 litters (7-8 embryos per litter) were dissected out and digested at 37°C for 3 min using TrypLE Express (Thermo Fisher Scientific). For E9.5 and E10.5 stages, posterior half of the embryo was dissected out and digested with trypsin solution (0.25% trypsin, 10% chick serum (both from Life Technologies), 1.27mM EDTA (Sigma)). Litters were pooled for stages up to and including E11.5 to obtain more material. Enzymatic digestion was neutralized with DMEM/F-12 (Gibco) supplemented with 15% fetal bovine serum (Gibco) after manual dissociation by gentle pipetting. The cells were spun down by centrifugation and resuspended in 0.1% BSA PBS. GFP-positive cells were isolated using an Aria Fusion (BD Bioscience) flow cytometer and sorted into ice-cold PBS. All preimplantation embryos were prepared by IVF. Collection of P6 and MII oocytes, and fertilized embryos was performed based on previous reports. Briefly, P6 oocytes were collected from C57BL/6J strain female mice at P6. The ovaries were dissected well with a microscissors before being incubated in 0.05% collagenase (gibco) dissolved in Dulbecco's modified Eagle's medium-F12 (DMEM/F12; Invitrogen) supplemented with 4 mg/ml bovine serum albumin (BSA; SIGMA), 80 µg/ml Kanamycin (SIGMA), with frequent pipetting. After 30 min, the oocytes were picked up using mouth pipette and transferred to PBS solution for washing. To select the oocytes at primordial follicle stage, the size of the oocytes was measured by an imaging software system (Octax EyeWare, Octax) and the 20-30 µm of oocytes were collected for further experiments. MII oocytes were collected from C57BL/6J female mice that were superovulated with serum gonadotropin (PMSG-Intervet; MSD) and human chorionic gonadotropin (hCG; CHORULON) at 46- to 52-h intervals. Spermatozoa were collected from the cauda epididymides of C57BL/6J male mice. The sperm suspension was preincubated in human tubal fluid (HTF) medium for 1.5 h at 37 °C under 5% CO2 in humidified air. The sperm suspension was added to the oocyte cultures, and morphologically normally fertilized oocytes were collected 2 h after insemination. Fertilized embryos were cultured in potassium simplex optimized medium (KSOM) at 37 °C under 5% CO2 in humidified air. Total RNA was isolated from the samples using the DNA/RNA Duet Kit miniprep kit (Zymo Research, USA). 5-20 ng of total RNA isolated from each sample was used for SLIC-CAGE TSS mapping. Briefly, 5 μg of the carrier RNA mix were added to each sample prior to reverse transcription, followed by the cap-trapping steps designed to isolate capped RNA polymerase II transcripts. The carrier was degraded from the final library prior to sequencing using homing endonucleases. The target library derived from the sample RNA polymerase II transcripts was PCR-amplified and purified using AMPure beads (Beckman Coulter) to remove short PCR artefacts (< 200bp, size selection using 0.8 x AMPure beads to sample ratio).