GSM5856509: week_7.2; Homo sapiens; RNA-Seq
Human pre-natal tissue was obtained from clinical routine abortions with oral and written consent from the patient. The acquisition of human pre-natal tissue was approved by the Swedish Ethical Research Authority and the National Board of Health and Welfare (ethical reference is 2018/769-31) for research purposes. The material was donated for a general research purpose especially with the focus on neuronal and nervous system related cell types. Measurement of crown-rump-length (CRL) and anatomical landmarks were used to determine the post-conception age. The tissue was received in ice-cold PBS and enzymatically digested to obtain the single cell suspension. For this, tissue cut into smaller pieces was incubated with 500 ul 0.05% Trypsin/0.02% EDTA (Sigma, 59417-C) for 15 min at 37°C with gentle swirling every 5 min. Gentle pipetting up and down was used to mechanically dissociate bigger pieces if any. 500 ul 10% FBS in PBS was added to cell suspension and the cells were pelleted at 500 rcf for 5 min at 4°C. Cells were washed two times with 1000 uL PBS, passed though the 35 um cell strainer (Falcon, 352235), pelleted at 500 rcf for 5 min at 4°C and re-suspended in 100 ul 0.04% BSA in PBS. Ice-cold methanol (400 uL was added for fixation of the cells and the cells were stored at -80°C. For the preparation of the library the cells were brought to +4°C and pelleted at 1000 rcf for 10 min at 4°C. Cell pellet was re-suspended in 500 uL of rehydration buffer (1X DPBS (Gibco 14190144) containing 1.0% BSA (Sigma, B4287) and 0.5U/μl RNase Out (ThermoFisher Scientific, 10777019)) followed by two washes with 500 uL of rehydration buffer. The rehydrated cell suspension was sorted to remove debris with BD FACS Aria Fusion instrument (BD Biosciences, San Jose, CA) equipped with 100 um nozzle. After FACS cells were pelleted to obtain concentrated cell suspension with 700-1200 cells/uL. 10x Genomics Chromium Single Cell 3ʹ protocol for Reagent Kits v3 was used for library preparation aiming to recover 5000-8000 cells.