Name: Sample 14
Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: RANDOM
Layout: SINGLE
Construction protocol: total 8 does were weighed and premedicated with ketamine 50 mg/kg kg (Ketamine 1000 CEVA; CEVA Sante Animale, Brussels, Belgium), xylazine 6 mg/kg (Vexylan; CEVA Sante Animale), and Buprenorphine 0.03 mg/kg (Vetergesic; Reckitt Benckiser Healthcare, Brussels, Belgium), all injected intramuscularly. General anesthesia was maintained using a face mask with isoflurane 1.5% (Isoba Vet; Abbott Laboratories Ltd., Queenborough, Kent, UK) in oxygen at 1 L/min. Maternal heart rate and oxygen saturation were monitored with a pulse oxymeter (Nellcor N-20P; Nellcor Inc., Haasrode, Belgium). Body temperature was maintained by a heating pad. The doe was placed in the supine position, the abdominal wall was shaved and disinfected with povidone iodine (Isobetadine; Asta Medica, Brussels, Belgium) and draped in a sterile fashion. Throughout all surgical procedures aseptic conditions were maintained. In every doe we operated on two fetuses each in the middle of the left or right uterine horn. First diaphragmatic hernia (DH) was created at 23 days' GA. A second operation was performed on these DH fetuses at 28 days' GA. At that time, one fetus underwent tracheal occlusion (TO group), whereas another underwent a sham operation (DH group), i.e. hysterotomy, fetal neck exposure, tracheal dissection without ligation, and skin closure. The latter is performed to exclude the effects of a fetal surgical procedure per se. At 30 days' GA, the does were euthanized and all operated fetuses and 1 control nonoperated littermate (WT) were harvested by cesarean section to obtain nonventilated lungs. total 8 does were weighed and premedicated with ketamine 50 mg/kg kg (Ketamine 1000 CEVA; CEVA Sante Animale, Brussels, Belgium), xylazine 6 mg/kg (Vexylan; CEVA Sante Animale), and Buprenorphine 0.03 mg/kg (Vetergesic; Reckitt Benckiser Healthcare, Brussels, Belgium), all injected intramuscularly. General anesthesia was maintained using a face mask with isoflurane 1.5% (Isoba Vet; Abbott Laboratories Ltd., Queenborough, Kent, UK) in oxygen at 1 L/min. Maternal heart rate and oxygen saturation were monitored with a pulse oxymeter (Nellcor N-20P; Nellcor Inc., Haasrode, Belgium). Body temperature was maintained by a heating pad. The doe was placed in the supine position, the abdominal wall was shaved and disinfected with povidone iodine (Isobetadine; Asta Medica, Brussels, Belgium) and draped in a sterile fashion. Throughout all surgical procedures aseptic conditions were maintained. In every doe we operated on two fetuses each in the middle of the left or right uterine horn. First diaphragmatic hernia (DH) was created at 23 days' GA. A second operation was performed on these DH fetuses at 28 days' GA. At that time, one fetus underwent tracheal occlusion (TO group), whereas another underwent a sham operation (DH group), i.e. hysterotomy, fetal neck exposure, tracheal dissection without ligation, and skin closure. The latter is performed to exclude the effects of a fetal surgical procedure per se. At 30 days' GA, the does were euthanized and all operated fetuses and 1 control nonoperated littermate (WT) were harvested by cesarean section to obtain nonventilated lungs. RNA isolation was performed on the whole left lung samples within 4hrs of harvesting using the RNeasy mini kit (Qiagen Benelux B.V., Venlo NL ). Tissue lysis and homogenization was performed in 1200ul Buffer RLT using the TissueLyser system (Qiagen Benelux B.V., Venlo NL). One ug of total RNA was used as input material for sequencing library preparation which was performed with the TruSeq RNA Library Preparation Kit (Illumina) according to the manufacturers protocol. Fragmentation was performed for 6 mins. 8 PCR cycles were used for PCR enrichment step. Samples were indexed to allow for multiplexing.