title: Single cell RNA-Seq of post mortem COVID19 cases from Malawi (lung, nasal & blood)
design: Single cell RNA-Seq of post mortem COVID19 cases from Malawi (lung, nasal & blood)
library_construction_protocol: We used minimally invasive sampling to conduct autopsies which large-bore needle biopsies of organ samples rather than full autopsy. Being more culturally acceptable, MITS is widely used to determine cause of death in paediatric studies, showing good concordance with full autopsy. From our ongoing paediatric MITs studies in Malawi, we adapted protocols for adult patients with Covid19 to obtain tissue suitable for single cell RNA-sequencing (scRNA-sequencing) and IMC. Based on CHAMPS but with adaptations. In particular a larger calibre needle (12 gauge) was used for biopsies to obtain larger tissue samples. From each lung, samples were taken from lower middle and upper zones from a single entry point, angling the needle to sample different areas.   The study protocol was approved by the National Health Scientific Research Committee (NHSRC) in Malawi (Protocol number 07/09/1913) and by the Medical Veterinary Life Sciences ethics committee in Glasgow (Protocol number 200190041). Informed consent was taken from the families of deceased patients. We underwent a full sensitisation process for the study with all staff on the recruiting wards in our hospital to discuss the study and consider the best way of sensitively conducting recruitment and informed consent this work was led by two social scientists (LS, DN) one with special interest in medical ethics (DN).  Lung samples were dissociated both from fresh samples and from slow frozen samples that had been stored in liquid nitrogen. Slow frozen cells were defrosted using a defrosting protocol described previously (Delorey). Fresh or defrosted frozen cells were then dissociated using a similar method to Travaglini et al. Briefly cells were dissociated in a buffer containing 400g/mL of Liberase DL (Sigma), 32U/ml of DNAse I (Roche) and 1.5% BSA in PBS (without calcium and magnesium). The tissue was put in buffer (4 times weight:volume) in a GentleMACS C-tube (Miltenyi 130-096-334) minced with scissors and then run on a GentleMACS dissociator (130-093-235) on programme “C-lung 01_02”. Dissociation was achieved by warming tissue on an orbital shaker in a chamber at 37C for 30 mins and running “C-lung 01_02” twice more; once at 15 mins and once at 30mins. Enzyme was neutralised by diluting with 10ml of ice cold 20% FBS with 32u/ml of DNase and the sample was filtered through a 100M strainer (352360) and samples were subsequently kept on ice with all centrifuge and antibody incubation steps at 4c. Cells were pelleted by spinning at 300g for 5 mins and red cells removed by incubation with ACK buffer for 5mins. For frozen cells debris were removed using a debris removal solution (Miltenyi, 130-109-398) according to the manufacturers protocol. Single nuclei were prepared from snap frozen lung samples as described previously (REF). Briefly , Frozen lung tissue kept on dry ice/ liquid nitrogen until processing was added to a gentle MACS C-tube containing 2ml of freshly prepared nuclei extraction buffer which contained RNAse inhibitors; 0.2 U/µL RNaseIN Plus RNAse inhibitor (Promega) and 0.1 U/µL SUPERasin RNAse inhibitor (Thermofisher scientific). Dissociation was achieved by running the C-tube on GentleMACs dissociator on program ''m_spleen_01'' for 1 minute. The sample was then filtered using a 40 µM strainer. The C-tube and strainer were rinsed using a buffer containing 0.1% enzymatics RNAse inhibitor (Enzymatics). Sample was then pelleted by spinning at 500g for 10 minutes at 40C. Pellet was then resuspended in 500ul of 1xST without RNAse Inhibitor, resuspension volume varied between 0.2-3ml depending on input amount of tissue. Volume adjustment was done after assessing the concentration of nuclei/debris under a microscope to ease counting. The sample was then filtered using 35µM strainer, a 10 µL volume was loaded on haemocytometer for counting without trypan blue. 10x Genomics 3 prime version 3 chemistry was used for all samples and the library was generated according to the manufacturers protocol.