title: Illumina HiSeq 4000 sequencing; Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohn's disease
design: Single-cell sequencing of developing human gut reveals transcriptional links to childhood Crohn's disease
library_construction_protocol: First trimester human fetal tissue was collected from patients undergoing elective termination of pregnancy. Patients gave informed consent as part of the ethically approved research study (REC-96/085). Fetal age (post conception weeks, PCW) was estimated using the independent measurement of the crown rump length (CRL), using the formula PCW (days) = 0.9022 × CRL (mm) + 27.372. Paediatric biopsies or fetal tissue sections were immediately rinsed twice with Hank's Balanced Salt Solution (HBSS) medium (Sigma-Aldrich) and digested in HBSS medium containing 1.07 Wünsch units/ml of Liberase DH (Roche) and 600 IU of Hyaluronidase (Calbiochem) on a shaking platform (750 rpm) at 37oC for up to 30 min. The tissue was gently homogenised using a P1000 pipette every 15 mins. A single-cell suspension was then passed through a 40 μm cell strainer to remove undigested tissue. Cells were spun down at 400 g at 4oC for 5 min and the pelleted cells were washed in DMEM/F12 three times using centrifugation.  Human intestinal mucosal biopsies were obtained from patients undergoing colonoscopy at Addenbrooke's Hospital, Cambridge, UK. All patients gave informed consent for extra biopsy samples to be taken for research use when undergoing elective colonoscopy (REC 17/EE/0265). Patients were then included if, after macroscopic visualization and histological analysis, they were diagnosed as either having Crohn's disease, or being without an inflammatory diagnosis (control). Paediatric biopsies or fetal tissue sections were immediately rinsed twice with Hank's Balanced Salt Solution (HBSS) medium (Sigma-Aldrich) and digested in HBSS medium containing 1.07 Wünsch units/ml of Liberase DH (Roche) and 600 IU of Hyaluronidase (Calbiochem) on a shaking platform (750 rpm) at 37oC for up to 30 min. The tissue was gently homogenised using a P1000 pipette every 15 mins. A single-cell suspension was then passed through a 40 μm cell strainer to remove undigested tissue. Cells were spun down at 400 g at 4oC for 5 min and the pelleted cells were washed in DMEM/F12 three times using centrifugation. For enrichment, single cells were suspended in MACS modified solution (PBS with 0.5% BSA, 2 mM EDTA and 100 IU/mL DNaseII) and stained with EPCAM (CD326) magnetic microbeads (MiltenyiBiotec) according to the manufacturer's protocol. Enrichment was performed using an autoMACS Pro Separator. Both fetal and paediatric single-cell suspensions were carried forward into single-cell sequencing only if the viability was >60%  10x Genomics Chromium Single Cell 3' Reagent Kit (v2 chemistry) As per Chromium 10x v2 protocol