First and second strand cDNA synthesis and pre-amplification

SINGLE CELL RNASEQ of malaria parasites

*Be paranoid about how a nuclease could get in the sample at all times

*Work only with dedicated fresh reagents

*Maximise work in the MSC

*Use RNAseZAP on all surfaces you work on

*Decon hoods with UV light for an hour before working in them

*Use nuclease-free water for molecular biology (not DEPC-treated)

*Clean sorter with bleach (1%) before experiment and preferably every night after use

SAMPLE PREPARATION

It is important that parasites are as pure as possible (maximum proportion of sortable cells). Therefore purifying the parasites before fixing and sorting is best.

*Schizonts

• Smear the culture
• To make up 63% isotonic Percoll: add 10 ml sterile 10X PBS to 90 ml neat Percoll (Percoll Plus 17-5445-01, GE Healthcare). Then add to this 42.8 ml Serum-Free RPMI. Store at 4 °C
• To enrich schizonts: take a parasite culture in RPMI containing quantities of mature, segmented schizonts. Pellet the culture (1100 x g, 5 min). Remove the supernatant and resuspend the pellet to a haematocrit of about 30-40% in RPMI + Albumax (ie up to 2 ml packed cells in a total of 5 ml)
• Using a 5 mL pipette, carefully layer up to 7.5 ml of the parasite suspension onto 10 ml cushions of 63% Percoll (at room temperature) in 15mL (better) or 50 mL Falcon tubes. Centrifuge at 1,300 x g, 11 min, at RT, w/o break.
• Carefully remove and keep the dark band (enriched for mature schizonts) at the interface between the RPMI and the Percoll. Transfer the cells to a fresh 50 ml tube containing about 30 ml SF RPMI. Pellet the schizonts at 900 x g for 5 min (so far this has been done on GFP schizonts without staining)

*Rings

• Smear the culture
• Pass the culture on MACs column in SF medium to deplete possible contaminating late stages (only for cultured parasites)
• Incubate the culture on ice with 1µg/ml Hoechst 33342 for 10 minutes in SF medium
• Spin the culture and wash once in 1X Dulbecco's PBS (DPBS)
• Resuspend in 1 ml of DPBS and transfer to RNAse free 1.5 mL Eppendorf
• Spin at 2000g for 1 min at 4 degrees
• Add 1 ml of 0.15% Saponin (in PBS) and incubate on ice for 2 minutes
• Spin at 2000g for 1 min at 4 degrees and wash with 1 ml DPBS, repeat once
• Parasites are ready to sort, no need to fix
• Gating should be determined by control naïve erythrocytes prepared in the same way

SAMPLE FIXATION

• Take pelleted parasites to the hood and resuspend parasites in approx. 50 µl of supernatant, use 5 µl to smear the purified parasites
• Resuspend parasites in 500 µl of RNALater
• Incubate for 5 minutes
• Add 7 volumes (3.5ml) of DPBS and transfer to FACs tube
• Keep samples on ice and try to minimize the amount of time between fixation and sorting

PLATE PREPARATION

• Prepare plates for sorting on the day of the experiment, concomitantly with parasite prep or before
• Dilute 0.8% Triton-X from a 10% stock prepare with nuclease-free water (AM9939) in a 50 ml falcon tube
• Put the tube under UV light in the cross-linker at 2000 joules/cm for 30 minutes
• Use Abgene (10009092) plates only
• Prepare a master mix of the following in the MSC

• Distribute 4 µl of the mix in each of the plate wells
• Cover the plate with a sterile film
• Spin briefly at 1000 rpm
• Keep the plates on ice

SORTING

• Work in one of the Hooded sorters (Influx has shown better results and has the possibility of index sorting so is preferred)
• Decon surfaces in the hood with RNAseZap
• Use the refrigerated plate holder
• Include 0 and 100 controls on plate
• Once the sort is complete, seal the plate in the hood.
• Spin briefly at 1000 rpm in refrigerated centrifuge and transfer to a bed of dry ice immediately
• Make note of map, record plate order and time it took to sort
• Get sort data (gates…) and index files
• Some early data indicated that plates that were processed directly were better. Otherwise keep plates at -80 °C

REVERSE TRANSCRIPTION AND PCR

• Transport samples on dry ice to a clean PCR hood which has not been used for generating post-pcr NexteraXT libraries and has been cleaned thoroughly with RNAseZap.
• Prepare RT mastermix for smart-seq2 (in order below), invert tube and spin briefly then store on ice until use.

• Place plate on thermocycler and run the denaturing step at 72 °C  3 min. Immediately afterwards spin down plate for 10 s 1500 rpm and place either on cooled metal block for 96 well plates.
• Dispense 5.7 µl of RT mastermix into each well using the Eppendorf Multipipette Xstream with combitip. Angle the tip towards the side of the well at the very top. To reduce the possibility of cross-contamination pipette into empty wells or negative control wells first, then single cell wells, then multi-cell wells.
• Place plate sealer (AB micro Amp Clean Adhesive Seal, 4306311) and ensure a tight seal, particularly at the edges of the plate.
• Spin down your plate and run the following programme on thermocycler to reverse transcribe (takes 2hrs 10 mins): Step 1: 42 °C 1.5hr

        Step 2: 42 °C 2 min

        Step 3: 50 °C 2 min

        Step 4: Goto Step 2 x9

        Step 5: 70 °C 15 min

        Step 6: 4 °C forever

• Proceed to smartseq-2 PCR step ideally on the same day (as it is a long PCR that takes over 3hrs can leave it overnight).
• Add 15 µl of PCR mastermix using a multichannel pipette and individual tips:

• Place plate sealer and ensure a tight seal, particularly at the edges of the plate. Spin down your plate and run the following programme on thermocycler for PCR:

        Step 1: 98 °C 3 min

        Step 2: 98 °C 20 s

        Step 3: 67 °C 15 s

        Step 4: 72 °C 6 min

        Step 5: Goto Step 2 x24 (n.b. will be testing reduced no. cycles with optimised protocol but previous attempts with 20 and 25 cycles have been unsuccessful)

        Step 5: 72 °C 5 min

        Step 6: 4 °C forever

• Do not unseal the plate in the clean zone used for previous reactions - take to a different lab.
• Warm Agencourt Ampure XP beads (Beckman Coulter, A63881) for 20 min until room temperature. Use at 1x ratio for clean-up (25 µl per sample).
  ○ Add beads (use multichannel and clean boat) and allow to bind at room temperature for 6 min
  ○ Place on 96 well magnet and allow to settle for 5 min
  ○ Remove 50 µl supernatant using multichannel.
  ○ Without removing the plate from the magnet add 200 µl 80% ethanol in nuclease-free water (make fresh each day) and remove. Do not attempt to resuspend beads.
  ○ Repeat ethanol wash
  ○ Carefully remove any remaining ethanol from wells.
  ○ Allow the beads to dry for 8 min.
  ○ Remove plate from magnet and using a multi-channel resuspend beads in 10 µl nuclease-free water (x6 up and down depending on how dry the beads are).
  ○ Place on magnet and leave for at least 5 min. Remove eluted amplified cDNA (being careful to not transfer beads) to new plate and seal.
• Store all plates at -20 °C to reduce possibility of evaporation. 
  *Alternatively use the Zehpr robot for 96 well clean up, however restriction on elute volume is 20 µl.
• Store all plates at -20 °C to reduce possibility of evaporation.

INITIAL QC OF THE SAMPLES

*Agilent

Use the high sensitivity DNA kit to assess the amplified cDNA quantity and distribution on the Agilent bioanalyser. Follow the kit instructions. Kit needs to be warmed to room temperature 30 mins before use. This QC will use 1 µl of each sample. Ideally place plate or samples on magnet prior to taking 1 µl as any stray Ampure beads will cause spikey traces. 
LIBRARY PREP

NexteraXT (quarter reactions)

• First make index plate, add 10 µl of each index across the plate for matrix. Seal well. This plate should be sufficient for 4 reactions (1/4 reactions). This can be stored at -20°C. 4 plates can be sequenced concurrently by using indices A-D.
• Make one plate at a time

Mix

• Distribute 3.75 µl in clean empty plate Use an 8 well strip to pipette from across the plate with a multichannel.
• Add 1.25 µl of the clean-uped cDNA sample.
• Seal, briefly spin and incubate at 55°C for 10 min. Let the block reach 10°C.
• Spin briefly then add 1.25 µl NT buffer per well, seal and briefly spin.
• Add 3.75 µl NPM per sample, seal and briefly spin.
• Add 2.5 µl index mix.
• Run the NexteraXT PCR (12 cycles).

Samples can be pooled for 0.8x (12 µl l per reaction) bead clean up, eluted in 10 µl for each sample.

SEQUENCING

Samples cab be submitted in 384-plex on a Hiseq lane using 4 sets of dual indices from Illumina(A,B,C,D)