title: GSM4790409: ya93flow480: HUVEC under flow 48 hr rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Human umbilical cords were collected under Institutional Review Board (UCLA IRB#16-001694) at time of the delivery and processed 2-4 hours from time of birth. The umbilical vein was cannulated with a 18G animal feeding needle with a blunt tip in the direction of oxygenated blood flow from the placenta to the fetus. The umbilical vein was serially washed with 20 mL of phosphate-buffered saline (PBS) 2x. Subsequently, for ex-vivo samples 1mL of RLT from RNeasy Micro Kit (Qiagen, Germantown, MD), was flushed through closed circuit and re-aspirated with the distal end of the umbilical cord clamped and stored in -80-degrees C until all RNA was ready to be extraction. The length of time to obtained cells was approximately 30-60 minutes from cord clamping at delivery. For the in-vitro samples, after PBS wash, 8 mL of collagenase (210 IU, Worthington Biochemical, Lakewood, NJ) was flushed into the umbilical vein and it was incubated at 37-degrees C for 20-minutes. The flushed cells were subsequently collected and collagenase was inactivated with the addition of equivalent volume 10% feta bovine serum (FBS) with MCDB 131 media (VEC Technologies, Rensselaer, NY). The cells were pelleted, resuspended in media and plated. After cells became confluent they were passaged using 1x Trypsin (Corning, Manassas, VA) and cells were collected with RLT for RNA extraction in early passage (passage 2-3) or late passage (passage 7-8).  Subsequently all RNA was extracted concomitantly using RNeasy Micro Kit (Qiagen, Germantown, MD). Contamination with genomic DNA was eliminated with incubation of DNase I at room temperature. Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA) was used to assess RNA integrity and Qubit (Invitrogen, Carlsbad, CA) was used for RNA concentration and purity. RNA libraries were prepared for sequencing using standard Illumina protocols