title: f-KS1-B
organism: Homo sapiens
description: Protocols: Reprogramming of fibroblasts with episomal plasmids was performed as previously reported(Okita et al., 2011) with some modifications. Briefly, 6x105 cells were electroporated with 1 µg of each plasmid; pCXLE-hOCT3/4-shp53, pCXLE-hSK, and pCXLE-hUL (gift from Dr. Shinya Yamanaka), using the 100 μl tip Neon System (Life Technologies) with 1650 V, 30 ms and 1 pulse settings. Transfected cells were plated onto non-coated cell culture plates with HDF medium for six days in 5 % CO2, 37°C. Cells were collected with 0.25 % Trypsin (Life Technologies) and plated onto 10 μg/ml LN521 (Biolamina) coated plates or onto hESC-qualified Matrigel (BD Biosciences) coated plates with a density of 5x103 cells/cm2 and transferred to low oxygen conditions (5 % O2, 5 % CO2) at 37°C. The following day, HDF medium was replaced by Nutristem hPSC XF Medium (Biological Industries) with following medium changes every 2-3 days. Cell colonies were manually picked onto LN521 coated plates 23-40 days after transfection and maintained in Nutristem hPSC XF Medium throughout the cultures. Upon confluency, cells were passaged as single cells using 1x TrypLE Select (Life Technologies). HESC line HS980, 46,XX(Rodin et al., 2014)was used in this study with the approval of the Stockholm Regional Ethics Board (Dnr. 454/02). RNA was extracted using the RNeasy Mini Kit (Qiagen) with on-column DNaseI treatment (Qiagen) according to the manufacturer's protocol from three different passages of f-HM1 (p8-10), f-HM2 (p7, p10-11), f-KS1 (p5, p8-9), f-KS2 (p5, p7-8), f-HF1 (p12-14), ips-HM1 (p13-15), ips-HM2 (p11-13), ips-KS1 (p13-15), ips-KS2 (p11-13), ips-HF1 (p15-17), and HS980 (p23-25). Library preparation and sequencing was performed at the Science for Life Laboratory, Sweden. Briefly, libraries were made using Illumina TruSeq® Stranded mRNA with poly-A selection.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-10-05T04:02:43Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-10-25T16:28:03Z
attributes.tag: External Id
attributes.value: SAMEA5046563
attributes.tag: INSDC center name
attributes.value: Department for Clinical Science, Intervention and Technology, Division of Obstetrics and Gynecology, K57, Karolinska Institutet
attributes.tag: INSDC first public
attributes.value: 2019-10-05T04:02:43Z
attributes.tag: INSDC last update
attributes.value: 2018-10-25T16:28:03Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7340:f-KS1-B
attributes.tag: age
attributes.value: 31
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: fibroblast of dermis
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: Klinefelter's syndrome
attributes.tag: growth condition
attributes.value: passage 5, 8-9
attributes.tag: individual
attributes.value: 4
attributes.tag: karyotype
attributes.value: 47,XXY
attributes.tag: organism part
attributes.value: skin
attributes.tag: sample name
attributes.value: E-MTAB-7340:f-KS1-B
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: male